Anti-CCL27 antibodies

ABSTRACT

Novel CC chemokines from human, reagents related thereto including purified proteins, specific antibodies and nucleic acids encoding these chemokines are provided. Also provided are methods of making and using said reagents and diagnostic kits.

The present application is a division of U.S. application Ser. No.10/146,496; filed May 15, 2002 which is a continuation-in-part of U.S.application Ser. No. 08/978,964; filed Nov. 26, 1997, which claimsbenefit of U.S. provisional patent application 60/031,805, filed Nov.27, 1996; and 60/032,606, filed Dec. 5, 1996; and 60/063,259, filed onOct. 24, 1997; each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention contemplates compositions related to proteinswhich function in controlling development, differentiation, trafficking,and physiology of mammalian cells, e.g., cells of a mammalian immunesystem. In particular, it provides proteins which regulate or evidencedevelopment, differentiation, and function of various cell types,including hematopoietic cells.

BACKGROUND OF THE INVENTION

The circulating component of the mammalian circulatory system comprisesvarious cell types, including red and white blood cells of the erythroidand myeloid cell lineages. See, e.g., Rapaport (1987) Introduction toHematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood:Textbook of Hematology, Little, Brown and Co., Boston, Mass.; and Paul(ed.) (1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork.” Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which should lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system and other disorders.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, anddifferentiation of the pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. These interactions between the cellularcomponents are necessary for a healthy immune response. These differentcellular lineages often respond in a different manner when lymphokinesare administered in conjunction with other agents.

The chemokines are a large and diverse superfamily of proteins. Thesuperfamily is subdivided into two classical branches, based uponwhether the first two cysteines in the chemokine motif are adjacent(termed the “C—C” branch), or spaced by an intervening residue(“C—X—C”). A more recently identified branch of chemokines lacks twocysteines in the corresponding motif, and is represented by thechemokines known as lymphotactins. Another recently identified branchhas three intervening residues between the two cysteines, e.g., CX3Cchemokines. See, e.g., Schall and Bacon (1994) Current Opinion inImmunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &Immunol. 109:97-109.

Many factors have been identified which influence the differentiationprocess of precursor cells, or regulate the physiology or migrationproperties of specific cell types. These observations indicate thatother factors exist whose functions in immune function were heretoforeunrecognized. These factors provide for biological activities whosespectra of effects may be distinct from known differentiation oractivation factors. The absence of knowledge about the structural,biological, and physiological properties of the regulatory factors whichregulate cell physiology in vivo prevents the modulation of the effectsof such factors. Thus, medical conditions where regulation of thedevelopment or physiology of relevant cells is required remainsunmanageable.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the alignment of protein sequences: Human MCP-1 (SEQ ID NO:9) is GenBank Accession number S71513; human MIP-3α (SEQ ID NO: 10) isGenBank Accession number U77035; the hCCL28 sequence is at SEQ ID NO: 2;the hCCL27 sequence is at SEQ ID NO: 6; the mCCL27 sequence is at SEQ IDNO: 8.

SUMMARY OF THE INVENTION

The present invention reveals the existence of previously unknownchemokine-motif containing molecules which are hereby designated CCL28(previously known as: DVic-1; MEC; and PLACC) and CCL27 (previouslyknown as CTACK; DGWCC; Eskine; ILC; and ALP). Based on sequence analysisof the chemokine protein sequences described below, it is apparent thatthe CCL27 and CCL28 belong to the CC chemokine family.

The present invention provides an isolated or purified polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 6, or an antigenic fragment thereof. Also provided is anisolated and purified polynucleotide having a sequence which iscomplementary to the polynucleotide of SEQ ID NO: 2 or SEQ ID NO: 6. Inanother embodiment, the polynucleotide can be detectably labeled.

The present invention further provides a method of detection comprising:a) hybridizing the polynucleotide of claim 3 to a sample containingnucleic acids under conditions suitable for the formation of ahybridization complex; and b) detecting the hybridization complex. Inanother embodiment, the polynucleotide is amplified prior tohybridization.

The present invention encompasses an isolated or purified polynucleotidewhich hybridizes under stringent conditions to the polynucleotide of SEQID NO:2 or SEQ ID NO:6. Also encompassed is an expression vectorcomprising the polynucleotide SEQ ID NO: 2 or SEQ ID NO: 6, and a hostcell comprising the expression vector.

Also encompassed is a method of producing a polypeptide comprising: a)culturing the host cell of claim 8 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide formthe host cell culture.

The present invention provides a substantially purified polypeptidecomprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6 or anantigenic fragment thereof. In another embodiment the polypeptide has80% amino acid sequence identity to SEQ ID NO: 2 or SEQ ID NO: 6. Inanother embodiment the polypeptide has 90% identity to SEQ ID NO: 2 orSEQ ID NO: 6. Also encompassed is a pharmaceutical compositioncomprising the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6 inconjunction with a suitable carrier.

The present invention provides a substantially purified bindingcomposition which binds to the polypeptide of SEQ ID NO: 2 or SEQ ID NO:6.

In another embodiment the binding composition is an antibody. Theantibody can be: a) a polyclonal antibody; b) a monoclonal antibody; c)a single chain antibody; d) an Fab fragment; e) an Fv fragment; or f) ahumanized antibody. In another embodiment the antibody is 6 capable ofblocking the binding of SEQ ID NO: 2 or SEQ ID NO: 6 to a receptor. Alsoencompassed is a pharmaceutical composition comprising the antibody inconjunction with a suitable carrier.

The present invention provides a method of modulating physiology ordevelopment of a cell or tissue culture cells comprising exposing saidcell to an agonist or antagonist of SEQ ID NO: 2 or SEQ ID NO: 6. In afurther embodiment the antagonist is an antibody.

DETAILED DESCRIPTION

I. General

The present invention provides primate DNA sequences encoding proteinswhich exhibit structural properties or motifs characteristic of acytokine or chemokine. For a review of the chemokine family, see, e.g.,Lodi, et al. (1994) Science 263:1762-1767; Gronenborn and Clore (1991)Protein Engineering 4:263-269; Miller and Kranger (1992) Proc. Nat'lAcad. Sci. USA 89:2950-2954; Matsushima and Oppenheim (1989) Cytokine1:2-13; Stoeckle and Baker (1990) New Biol. 2:313-323; Oppenheim, et al.(1991) Ann. Rev. Immunol. 9:617-648; Schall (1991) Cytokine 3:165-183;and The Cytokine Handbook Academic Press, NY.

The new cytokines described herein are designated CCL28 and CCL27. Seethe Sequence Listing. The descriptions below are directed, for exemplarypurposes, to primate embodiments, e.g., human, but are likewiseapplicable to related embodiments from other, e.g., natural, mammaliansources, including rodent. These sources should include variousvertebrates, typically warm blooded animals, e.g., birds and mammals,particularly domestic animals, rodents, and primates. Comparison toother chemokines is provided in FIG. 1.

The chemokine proteins of this invention are defined in part by theirphysicochemical and biological properties. The biological properties ofthe chemokines described herein, e.g., CCL28 or CCL27, are defined, inpart, by their amino acid sequence, and mature size. They also shouldshare at least some biological properties with other similar chemokines.One of skill will readily recognize that some sequence variations may betolerated, e.g., conservative substitutions or positions remote from thecritical residues for receptor interaction or important tertiarystructure features, without altering significantly the biologicalactivity of the molecule. Conversely, non-conservative substitutions maybe adapted to delete selected functions.

These chemokines are present in specific tissue types, e.g., skintissues, and the interaction of the protein with a receptor will beimportant for mediating various aspects of cellular physiology ordevelopment. The cellular types which express message encoding CCL28 orCCL27 suggest that signals important in cell differentiation anddevelopment are mediated by them. See, e.g., Gilbert (1991)Developmental Biology (3d ed.) Sinauer Associates, Sunderland, Mass.;Browder, et al. (1991) Developmental Biology (3d ed.) Saunders,Philadelphia, Pa.; Russo, et al. (1992) Development: The MolecularGenetic Approach Springer-Verlag, New York, N.Y.; and Wilkins (1993)Genetic Analysis of Animal Development (2d ed.) Wiley-Liss, New York,N.Y. Moreover, CCL28 or CCL27 expression or responsiveness should serveas markers, e.g., to define certain cell subpopulations.

The new chemokines were discovered through searches and careful analysisof database sequences. The absence of sequences in available databasesstrongly suggests that the messages are rarely expressed, and/or at verylow levels. Such may reflect highly restricted cell expression and/orvery low levels in most cell types.

CCL27 is closely homologous to CCL28, as shown by the alignment of thesetwo cytokines (FIG. 1). The sequences of MCP-1 (SEQ ID NO:9) and MIP-3α(SEQ ID NO:10) are also shown. Human CCL27 (SEQ ID Nos. 5 and 6) andhuman CCL28 (SEQ ID Nos. 1 and 2) share a 49% nucleotide identity forthe coding sequence, and about 40% amino acid identity in the commonhomology region (Pan, et al. (2000) J. Immunol. 165:2943-2949; Wang, etal. (2000) J. Biol. Chem. 275:22313-22323). Both CCL27 and CCL28 bind tothe same receptor, CCR10, though CCL28 can also bind to anotherreceptor, CCR3 (Pan, et al. (2000) J. Immunol. 165:2943-2949).

Human CCL27 (SEQ ID NO:6) and murine CCL27 (SEQ ID NO:8) share a 78%amino acid similarity. The predicted mature human and murine CCL27polypeptides are 84% similar (Morales, et al. (1999) Proc. Natl. Acad.Sci. USA 96:14470-14475). Screening of mouse cDNA libraries alsoprovided a longer transcript (SEQ ID NO:11) and polypeptide (SEQ IDNO:12) of murine CCL28, where the extra amino acids appear to contributeto a longer leader sequence for SEQ ID NO:12.

Human CCL28 (SEQ ID NO:1 and 2) is a CC chemokine of 127 amino acids,with a predicted 22 amino acid N-terminal signal peptide. Murine CCL28is a CC chemokine of 130 amino acids. Human and mouse CCL28 share a 76%sequence identity in the polynucleotide coding region, and share 83identical amino acids (Wang, et al. (2000) J. Biol. Chem.275:22313-22323). Initial phases of this work revealed expressedsequence tags (ESTS) derived from human fetal heart (SEQ ID NO:3) andhuman osteoblast (SEQ ID NO:4) cDNA libraries that contained partiallength sequences for the open reading frame of CCL28 (Wang, et al.(2000) supra).

CCL28 and CCL27 have been shown to share a common receptor which hasbeen identified as CCR10 (a.k.a. GPR2; see, e.g., Homey, et al. (2000)J. Immunol. 164:3465-3470.). As noted below CCL27 is secreted bykeratinocytes and binds to CCR10, which has high expression levels on Tcells which express Cutaneous Lymphocyte-associated Antigen (CLA; see,e.g., Homey, et al. (2002) Nature Med. 8:157-165). CCL27 attracts CLA+memory T cells and further, allergen induced skin inflammation (see,e.g., Santamaria, et al. (1995) Intl. Arch. Allergy Immunol.107:359-362) was ameliorated with administration of anti-CCL27antibodies. Therefore agonists of the present invention are useful inthe treatment of infectious disorders, in particular, infections of theskin. Antagonists of the present invention are useful for the treatmentof inflammatory disorders, in particular, skin inflammatory disorders,including, but not limited to, psoraisis, atopic dermatitis, contactdermatitis, etc.

II. Definitions

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,including selenomethionine, as well as those amino acids that aremodified after incorporation into a polypeptide, e.g., hydroxyproline,O-phosphoserine, O-phosphotyrosine, γ-carboxyglutamate, and cystine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an α-carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by their one-letter symbols.

The term “binding composition” refers to molecules that bind withspecificity and selectivity to a CCL28 or CCL27 chemokine, e.g., in anantibody-antigen interaction. However, other compounds, e.g., receptorproteins, may also specifically and/or selectively associate with CCL28or CCL27 chemokines to the exclusion of other molecules. Typically, theassociation will be in a natural physiologically relevantprotein-protein interaction, either covalent or non-covalent, and mayinclude members of a multiprotein complex, including carrier compoundsor dimerization partners. The molecule may be a polymer, or chemicalreagent. No implication as to whether a CCL28 or CCL27 chemokine isnecessarily a convex shaped molecule, e.g., the ligand or the receptorof a ligand-receptor interaction, is necessarily represented, other thanwhether the interaction exhibits similar specificity, e.g., specificaffinity. A functional analog may be a ligand with structuralmodifications, or may be a wholly unrelated molecule, e.g., which has amolecular shape which interacts with the appropriate ligand bindingdeterminants. The ligands may serve as agonists or antagonists of thereceptor, see, e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's:The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press,Tarrytown, N.Y.

The term “binding agent:chemokine protein complex”, as used herein,refers to a complex of a binding agent and a chemokine, e.g., CCL28 orCCL27, protein that is formed by specific binding of the binding agentto the chemokine protein. Specific or selective binding of the bindingagent means that the binding agent has a specific binding site, e.g.,antigen binding site, that recognizes a site on the CCL28 chemokineprotein that is not shared in many other proteins. For example,antibodies raised to a CCL28 chemokine protein and recognizing anepitope on the chemokine protein are capable of forming a binding agent:CCL28 chemokine protein complex by specific and selective binding.Typically, the formation of a binding agent:CCL28 chemokine proteincomplex allows the measurement of CCL28 chemokine protein in a mixtureof other proteins and biologics. Likewise, the term “antibody:CCL27chemokine protein complex” refers to an embodiment in which the bindingagent, e.g., is the antigen binding portion from an antibody. Theantibody may be monoclonal, polyclonal, or a binding fragment of anantibody, e.g., an Fab or F(ab)2 fragment. The antibody will preferablybe a polyclonal antibody for cross-reactivity testing purposes.

As used herein, the term “biological activity” is used to describe,without limitation, metabolic, signaling, hormonal, developmental,embryological, proliferative, apoptotic, secretory, migratory, adhesive,neurological, pathological, inflammatory, and cancerous activities of acell, tissue, organ, or animal, a cultured cell or tissue, a perfusedtissue or organ, or animal sustained on life support. “Biologicalactivity” also includes the catalytic activity of enzymes in vivo andenzymes in the purified state, as well as changes in conformation inenzymes and other proteins.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes aconserved amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant.” Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. An example of a conservative substitution is theexchange of an amino acid in one of the following groups for anotheramino acid of the same group (see, e.g., U.S. Pat. No. 5,767,063; andKyte and Doolittle (1982) J. Mol. Biol. 157:105-132).

-   (1) Hydrophobic: Norleucine, lie, Val, Leu, Phe; Cys; or Met;-   (2) Neutral hydrophilic: Cys, Ser, Thr;-   (3) Acidic: Asp, Glu;-   (4) Basic: Asn, Gin, His, Lys, Arg;-   (5) Residues that influence chain orientation: Gly; Pro; and-   (6) Aromatic: Trp; Tyr; Phe.-   (7) Small amino acids: Gly, Ala, Ser.

A polypeptide “fragment”, “antigentic fragment”, “immunogenic fragment”or “segment”, as used herein, encompasses a stretch of contiguous aminoacid residues of at least about 8 amino acids, generally at least 10amino acids, more generally at least 12 amino acids, often at least 14amino acids, more often at least 16 amino acids, typically at least 18amino acids, more typically at least 20 amino acids, usually at least 22amino acids, more usually at least 24 amino acids, preferably at least26 amino acids, more preferably at least 28 amino acids, and, inparticularly preferred embodiments, at least about 30 or more aminoacids, e.g., 35, 40, 45, 50, 60, 70, 80, 100, 200, etc. The inventionfurther comprises proteins or polypeptides comprising a plurality ofsaid segments, e.g., distinct, non-overlapping, segments of a specifiedlength. Typically, the plurality will be at least two, more usually atleast three, and preferably 4, 5, 6, 7, or even more. While the lengthminima are provided, longer lengths, of various sizes, may beappropriate, e.g., one of length 7, and two of length 12. Those skilledin the are will understand that antigenic or immunogenic fragments willbe those use to raise binding composition, specifically antibodies.Features of one of the different polypeptides or proteins defined insuch a manner should not be taken to limit those of another of thepolypeptides or proteins of the invention.

“Homologous” nucleic acid sequences, when compared, exhibit significantsimilarity, or identity. The standards for homology in nucleic acids areeither measures for homology generally used in the art by sequencecomparison and/or phylogenetic relationship, or based upon hybridizationconditions. Hybridization conditions are described in greater detailbelow.

“Humanized antibody” means an antibody comprising an antigen-bindingregion of nonhuman origin, e.g., rodent, and at least a portion of animmunoglobin of human origin, e.g., a human framework region, a humanconstant region, or portion thereof (see, e.g., U.S. Pat. No.6,352,832).

“Immune disorder” is defined as any condition where cells of the immunesystem, or molecules generally associated with the immune system, e.g.,cytokines, chemokines, and receptors thereof, contribute to thesuffering of a patient or, in the alternative, are not functioningoptimally to maintain the health of a patient. Immune disorders includeinflammatory conditions, e.g., psoriasis, allergies, asthma, rheumatoidarthritis, and cancer, metastasis, and the angiogenesis of tumors.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single stranded or double-stranded form. Theterm encompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids. An “isolated” nucleic acid is a nucleic acid,e.g., an RNA, DNA, or a mixed polymer, which is substantially separatedfrom other biologic components which naturally accompany a nativesequence, e.g., proteins and flanking genomic sequences from theoriginating species. The term embraces a nucleic acid sequence which hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs,or analogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule. Anisolated nucleic acid will usually contain homogeneous nucleic acidmolecules, but will, in some embodiments, contain nucleic acids withminor sequence heterogeneity. This heterogeneity is typically found atthe polymer ends or portions not critical to a desired biologicalfunction or activity.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., (1991) NucleicAcids Res. 19:5081; Ohtsuka, et al. (1985) J. Biol. Chem. 260:2605-2608;Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleicacid may be used interchangeably with gene, cDNA, mRNA, oligonucleotide,and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “allelicvariants” and “splice variants.” Similarly, a particular protein encodedby a nucleic acid implicitly encompasses any protein encoded by anallelic variant and splice variant of that nucleic acid. “Splicevariants” are products of alternative splicing of mRNA. Aftertranscription, an mRNA may be spliced such that different (alternate)nucleic acid splice products encode different polypeptides. Mechanismsfor the production of splice variants vary, but include alternatesplicing of exons. Alternate polypeptides derived from the same nucleicacid by read-through transcription are also encompassed by thisdefinition. Any products of a splicing reaction, including recombinantforms of the splice products, are included in this definition. It willbe understood that inasmuch as natural allelic variations exist andoccur from individual to individual, as demonstrated by (an) amino aciddifference(s) in the overall sequence or by deletions, substitutions,insertions, inversions, or additions of one or more amino acids of saidsequences, the present invention is intended to embrace all of suchallelic variations of the two molecules involved.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

As used herein, the term “CCL28 chemokine protein” shall encompass, whenused in a protein context, a protein having amino acid sequences,particularly from the chemokine motif portions, shown in SEQ ID NO: 2,or a significant fragment unique to and/or characteristic of such aprotein, preferably a natural embodiment. Likewise for the human andmouse CCL27, and SEQ ID NO: 6 and 8. The invention also embraces apolypeptide which exhibits similar structure to such chemokines, e.g.,which interacts with CCL28 or CCL27 chemokine specific bindingcomponents. These binding components, e.g., antibodies, typically bindto either CCL28 or CCL27 chemokine with high affinity, e.g., at leastabout 100 nM, usually better than about 30 nM, preferably better thanabout 10 nM, and more preferably at better than about 3 nM.

The term “polypeptide” or “protein” as used herein includes asignificant fragment or segment of chemokine motif portion of, e.g., aCCL27 chemokine, and encompasses a stretch of amino acid residues of atleast about 8 amino acids, generally at least 10 amino acids, moregenerally at least 12 amino acids, often at least 14 amino acids, moreoften at least 16 amino acids, typically at least 18 amino acids, moretypically at least 20 amino acids, usually at least 22 amino acids, moreusually at least 24 amino acids, preferably at least 26 amino acids,more preferably at least 28 amino acids, and, in particularly preferredembodiments, at least about 30 or more amino acids, e.g., 35, 40, 45,50, 60, 70, 80, etc. The segments may have amino and carboxy termini,with appropriate lengths, e.g., starting, e.g., at residue 1, 2, 3,etc., and ending at, e.g., residue 95, 94, 93, 92, etc. The inventionencompasses proteins comprising a plurality of said segments.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, for example, products made by transforming cells with anynon-naturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site. Alternatively, it is performed tojoin together nucleic acid segments of desired functions to generate asingle genetic entity comprising a desired combination of functions notfound in the commonly available natural forms. Restriction enzymerecognition sites are often the target of such artificial manipulations,but other site specific targets, e.g., promoters, DNA replication sites,regulation sequences, control sequences, or other useful features may beincorporated by design. A similar concept is intended for a recombinant,e.g., fusion, polypeptide. Specifically included are synthetic nucleicacids which, by genetic code redundancy, encode polypeptides similar tofragments of these antigens, and fusions of sequences from variousdifferent species variants.

“Substantially pure” in a protein context typically means that theprotein is isolated from other contaminating proteins, nucleic acids,and other biologicals derived from the original source organism. Purity,or “isolation” may be assayed by standard methods, and will ordinarilybe at least about 50% pure, more ordinarily at least about 60% pure,generally at least about 70% pure, more generally at least about 80%pure, often at least about 85% pure, more often at least about 90% pure,preferably at least about 95% pure, more preferably at least about 98%pure, and in most preferred embodiments, at least 99% pure. Similarconcepts apply, e.g., to antibodies or nucleic acids.

“Substantial similarity” in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least 56%, more generally at least 59%,ordinarily at least 62%, more ordinarily at least 65%, often at least68%, more often at least 71%, typically at least 74%, more typically atleast 77%, usually at least 80%, more usually at least about 85%,preferably at least about 90%, more preferably at least about 95 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides. Alternatively, substantial similarity exists when thesegments will hybridize under selective hybridization conditions, to astrand, or its complement, typically using a sequence derived from SEQID NO: 1, 5, or 7. Typically, selective hybridization will occur whenthere is at least about 55% similarity over a stretch of at least about30 nucleotides, preferably at least about 65% over a stretch of at leastabout 25 nucleotides, more preferably at least about 75%, and mostpreferably at least about 90% over about 20 nucleotides. See Kanehisa(1984) Nuc. Acids Res. 12:203-213. The length of similarity comparison,as described, may be over longer stretches, and in certain embodimentswill be over a stretch of at least about 17 nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about40 nucleotides, preferably at least about 50 nucleotides, and morepreferably at least about 75 to 100 or more nucleotides, e.g., 150, 200,etc.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith and Waterman (1981) Adv.Appl. Math. 2:482, by the homology alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methodof Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms, as those provided bysoftware packages, such as MacVector 7.1® from Accelrys, Inc. (SanDiego, Calif.) and Vector NTI® Suite from InforMax, Inc. (Bethesda,Md.). See also, e.g., Needleham, et al., (1970) J. Mol. Biol.48:443-453; Sankoff, et al. (1983) Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.

“Stringent conditions”, in referring to homology or substantialsimilarity in the hybridization context, will be stringent combinedconditions of salt, temperature, organic solvents, and other parameters,typically those controlled in hybridization reactions. The combinationof parameters is more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370. A nucleic acid probe which binds to a target nucleic acidunder stringent conditions is specific for said target nucleic acid.Such a probe is typically more than 11 nucleotides in length, and issufficiently identical or complementary to a target nucleic acid overthe region specified by the sequence of the probe to bind the targetunder stringent hybridization conditions.

CCL27 chemokines from other mammalian species can be cloned and isolatedby cross-species hybridization of closely related species. See, e.g.,below. Similarity may be relatively low between distantly relatedspecies, and thus hybridization of relatively closely related species isadvisable. Alternatively, preparation of an antibody preparation whichexhibits less species specificity may be useful in expression cloningapproaches.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiological components. Thus, under designated immunoassay conditions,the specified antibodies bind to a particular protein and do notsignificantly bind other proteins present in the sample. Specificbinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. Forexample, antibodies raised to the CCL28 or CCL27 chemokine proteinimmunogen with the amino acid sequence depicted in SEQ ID NO: 2, or 6 or8, can be selected to obtain antibodies specifically immunoreactive withCCL27 chemokine proteins and not with other proteins. The antibodies maybe species specific, e.g., also recognizing polymorphic and splicing ordevelopmental variants.

“Therapeutically effective amount” of a therapeutic agent is defined asan amount of each active component of the pharmaceutical formulationthat is sufficient to show a meaningful patient benefit, i.e., to causea decrease in or amelioration of the symptoms of the condition beingtreated. When the pharmaceutical formulation comprises a diagnosticagent, “a therapeutically effective amount” is defined as an amount ofeach active component of the pharmaceutical formulation that issufficient to produce an image or other diagnostic parameter in thediagnostic system employed. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination of active ingredients, the termrefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. Effective amounts of the pharmaceutical formulation willvary according to factors such as the degree of susceptibility of theindividual, the age, sex, and weight of the individual, andidiosyncratic responses of the individual (see, e.g., U.S. Pat. No.5,888,530).

III. Nucleic Acids

CCL27 chemokine is exemplary of structurally and functionally relatedproteins. These soluble chemokine proteins will serve to transmitsignals between different cell types. The preferred embodiments, asdisclosed, will be useful in standard procedures to isolate genes fromdifferent individuals or other species, e.g., warm blooded animals, suchas birds and mammals. Cross hybridization will allow isolation ofrelated genes encoding proteins from individuals, strains, or species. Anumber of different approaches are available to successfully isolate asuitable nucleic acid clone based upon the information provided herein.Southern blot hybridization studies can qualitatively determine thepresence of homologous genes in human, monkey, rat, mouse, dog, cat,cow, and rabbit genomes under specific hybridization conditions.

Complementary sequences will also be used as probes or primers. Basedupon identification of the likely amino terminus, other peptides shouldbe particularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

Techniques for nucleic acid manipulation of genes encoding CCL27chemokine proteins, such as subcloning nucleic acid sequences encodingpolypeptides into expression vectors, labeling probes, DNAhybridization, and the like are described generally in Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, which isincorporated herein by reference. This manual is hereinafter referred toas “Sambrook, et al.”

There are various methods of isolating DNA sequences encoding CCL27chemokine proteins. For example, DNA is isolated from a genomic or cDNAlibrary using labeled oligonucleotide probes having sequences identicalor complementary to the sequences disclosed herein. Full-length probesmay be used, or oligonucleotide probes may be generated by comparison ofthe sequences disclosed. Such probes can be used directly inhybridization assays to isolate DNA encoding CCL27 chemokine proteins,or probes can be designed for use in amplification techniques such asPCR, for the isolation of DNA encoding CCL27 chemokine proteins. Reversetranslation computer programs can also provide alternative nucleic acidsequences which encode the same proteins.

To prepare a cDNA library, mRNA is isolated from cells which expresses aCCL27 chemokine protein. cDNA is prepared from the mRNA and ligated intoa recombinant vector. The vector is transfected into a recombinant hostfor propagation, screening, and cloning. Methods for making andscreening cDNA libraries are well known. See Gubler and Hoffman (1983)Gene 25:263-269 and Sambrook, et al.

For a genomic library, the DNA can be extracted from tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation and cloned in bacteriophage lambda vectors. These vectorsand phage are packaged in vitro, as described in Sambrook, et al.Recombinant phage are analyzed by plaque hybridization as described inBenton and Davis (1977) Science 196:180-182. Colony hybridization iscarried out as generally described in e.g., Grunstein, et al. (1975)Proc. Natl. Acad. Sci. USA. 72:3961-3965.

DNA encoding a CCL27 chemokine protein can be identified in either cDNAor genomic libraries by its ability to hybridize with the nucleic acidprobes described herein, e.g., in colony or plaque hybridization assays.The corresponding DNA regions are isolated by standard methods familiarto those of skill in the art. See, e.g., Sambrook, et al.

Various methods of amplifying target sequences, such as the polymerasechain reaction, can also be used to prepare DNA encoding CCL27 chemokineproteins. Polymerase chain reaction (PCR) technology is used to amplifysuch nucleic acid sequences directly from mRNA, from cDNA, and fromgenomic libraries or cDNA libraries. The isolated sequences encodingCCL27 chemokine proteins may also be used as templates for PCRamplification.

Typically, in PCR techniques, oligonucleotide primers complementary totwo 5′ regions in the DNA region to be amplified are synthesized. Thepolymerase chain reaction is then carried out using the two primers. SeeInnis, et al. (eds.) (1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif. Primers can be selectedto amplify the entire regions encoding a full-length CCL27 chemokineprotein or to amplify smaller DNA segments as desired. Once such regionsare PCR-amplified, they can be sequenced and oligonucleotide probes canbe prepared from sequence obtained using standard techniques. Theseprobes can then be used to isolate DNA's encoding CCL27 chemokineproteins.

Oligonucleotides for use as probes are usually chemically synthesizedaccording to the solid phase phosphoramidite triester method firstdescribed by Beaucage and Carruthers (1983) Tetrahedron Lett.22(20):1859-1862, or using an automated synthesizer, as described inNeedham-VanDevanter, et al. (1984) Nucleic Acids Res. 12:6159-6168.Purification of oligonucleotides is performed e.g., by native acrylamidegel electrophoresis or by anion-exchange HPLC as described in Pearsonand Regnier (1983) J. Chrom. 255:137-149. The sequence of the syntheticoligonucleotide can be verified using, e.g., the chemical degradationmethod of Maxam and Gilbert in Grossman and Moldave (eds. 1980) Methodsin Enzymology 65:499-560 Academic Press, New York.

An isolated nucleic acid encoding a CCL27 chemokine protein wasidentified. The nucleotide sequence and corresponding open reading frameare provided in SEQ ID NO: 1 or 5 or 7

These CCL27 and CCL28 chemokines exhibit limited similarity to portionsof chemokines. See, e.g., Matsushima and Oppenheim (1989) Cytokine1:2-13; Oppenheim, et al. (1991) Ann. Rev. Immunol. 9:617-648; Schall(1991) Cytokine 3:165-183; and Gronenborn and Clore (1991) ProteinEngineering 4:263-269. Other features of comparison are apparent betweenthe CCL27 chemokine and chemokine families. See, e.g., Lodi, et al.(1994) Science 263:1762-1766. In particular, β-sheet and α-helixresidues can be determined using, e.g., RASMOL program, see Sayle andMilner-White (1995) TIBS 20:374-376; or Gronenberg, et al. (1991)Protein Engineering 4:263-269; and other structural features are definedin Lodi, et al. (1994) Science 263:1762-1767. These secondary andtertiary features assist in defining further the C, CC, CXC, and CX3Cstructural features, along with spacing of appropriate cysteineresidues.

This invention provides isolated DNA or fragments to encode a CCL28 orCCL27 chemokine protein. In addition, this invention provides isolatedor recombinant DNA which encodes a protein or polypeptide which iscapable of hybridizing under appropriate conditions, e.g., highstringency, with the DNA sequences described herein. Said biologicallyactive protein or polypeptide can be an intact ligand, or fragment, andhave an amino acid sequence as disclosed in SEQ ID NO: 2 or 6 or 8,particularly natural embodiments. Preferred embodiments will be fulllength natural sequences, from isolates, e.g., about 11,000 to 12,500daltons in size when unglycosylated, or fragments of at least about6,000 daltons, more preferably at least about 8,000 daltons. Inglycosylated form, the protein may exceed 12,500 daltons. Further, thisinvention contemplates the use of isolated or recombinant DNA, orfragments thereof, which encode proteins which are homologous to a CCL28or CCL27 chemokine protein or which were isolated using cDNA encoding aCCL28 or CCL27 chemokine protein as a probe. The isolated DNA can havethe respective regulatory sequences in the 5′ and 3′ flanks, e.g.,promoters, enhancers, poly-A addition signals, and others. Also embracedare methods for making expression vectors with these sequences, or formaking, e.g., expressing and purifying, protein products.

IV. Making CCL28, CCL27 Chemokines

DNAs which encode a CCL28 or CCL27 chemokine or fragments thereof can beobtained by chemical synthesis, screening cDNA libraries, or byscreening genomic libraries prepared from a wide variety of cell linesor tissue samples. Methods for doing so, or making expression vectorsare described herein.

These DNAs can be expressed in a wide variety of host cells for thesynthesis of a full-length protein or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies. Each CCL28 or CCL27 chemokine or itsfragments can be expressed in host cells that are transformed ortransfected with appropriate expression vectors. These molecules can besubstantially purified to be free of protein or cellular contaminants,other than those derived from the recombinant host, and therefore areparticularly useful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The antigen, e.g.,CCL27 chemokine, or portions thereof, may be expressed as fusions withother proteins or possessing an epitope tag.

Expression vectors are typically self-replicating DNA or RNA constructscontaining the desired antigen gene or its fragments, usually operablylinked to appropriate genetic control elements that are recognized in asuitable host cell. The specific type of control elements necessary toeffect expression will depend upon the eventual host cell used.Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently from thehost cell.

The vectors of this invention contain DNAs which encode a CCL28 or CCL27chemokine, or a fragment thereof, typically encoding, e.g., abiologically active polypeptide, or protein. The DNA can be under thecontrol of a viral promoter and can encode a selection marker. Thisinvention further contemplates use of such expression vectors which arecapable of expressing eukaryotic cDNA coding for a CCL28 or CCL27chemokine protein in a prokaryotic or eukaryotic host, where the vectoris compatible with the host and where the eukaryotic cDNA coding for theprotein is inserted into the vector such that growth of the hostcontaining the vector expresses the cDNA in question. Usually,expression vectors are designed for stable replication in their hostcells or for amplification to greatly increase the total number ofcopies of the desirable gene per cell. It is not always necessary torequire that an expression vector replicate in a host cell, e.g., it ispossible to effect transient expression of the protein or its fragmentsin various hosts using vectors that do not contain a replication originthat is recognized by the host cell. It is also possible to use vectorsthat cause integration of a CCL28 or CCL27 chemokine gene or itsfragments into the host DNA by recombination, or to integrate a promoterwhich controls expression of an endogenous gene.

Vectors, as used herein, contemplate plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector, but many other forms of vectors whichserve an equivalent function are suitable for use herein. See, e.g.,Pouwels, et al. (1985 and Supplements) Cloning Vectors: A LaboratoryManual Elsevier, N.Y.; and Rodriquez, et al. (eds.) (1988) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses Buttersworth, Boston,Mass.

Suitable host cells include prokaryotes, lower eukaryotes, and highereukaryotes. Prokaryotes include both gram negative and gram positiveorganisms, e.g., E. coli and B. subtilis. Lower eukaryotes includeyeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyosetlium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 or itsderivatives. Vectors that can be used to express CCL27 chemokines orCCL27 chemokine fragments include, but are not limited to, such vectorsas those containing the lac promoter (pUC-series); trp promoter(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters(pOTS); or hybrid promoters such as ptac (pDR540). See Brosius, et al.(1988) “Expression Vectors Employing Lambda-, trp-, lac-, andIpp-derived Promoters”, in Rodriguez and Denhardt (eds.) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses 10:205-236Buttersworth, Boston, Mass.

Lower eukaryotes, e.g., yeasts and Dictyosetlium, may be transformedwith CCL28 or CCL27 chemokine sequence containing vectors. For purposesof this invention, the most common lower eukaryotic host is the baker'syeast, Saccharomyces cerevisiae. It will be used generically torepresent lower eukaryotes although a number of other strains andspecies are also available. Yeast vectors typically consist of areplication origin (unless of the integrating type), a selection gene, apromoter, DNA encoding the desired protein or its fragments, andsequences for translation termination, polyadenylation, andtranscription termination. Suitable expression vectors for yeast includesuch constitutive promoters as 3-phosphoglycerate kinase and variousother glycolytic enzyme gene promoters or such inducible promoters asthe alcohol dehydrogenase 2 promoter or metallothionine promoter.Suitable vectors include derivatives of the following types:self-replicating low copy number (such as the YRp-series),self-replicating high copy number (such as the YEp-series); integratingtypes (such as the YIp-series), or mini-chromosomes (such as theYCp-series).

Higher eukaryotic tissue culture cells are typically the preferred hostcells for expression of the functionally active CCL28 or CCL27 chemokineprotein. In principle, many higher eukaryotic tissue culture cell linesmay be used, e.g., insect baculovirus expression systems, whether froman invertebrate or vertebrate source. However, mammalian cells arepreferred to achieve proper processing, both cotranslationally andposttranslationally. Transformation or transfection and propagation ofsuch cells is routine. Useful cell lines include HeLa cells, Chinesehamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insectcell lines, bird cell lines, and monkey (COS) cell lines. Expressionvectors for such cell lines usually include an origin of replication, apromoter, a translation initiation site, RNA splice sites (e.g., ifgenomic DNA is used), a polyadenylation site, and a transcriptiontermination site. These vectors also may contain a selection gene oramplification gene. Suitable expression vectors may be plasmids,viruses, or retroviruses carrying promoters derived, e.g., from suchsources as from adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pcDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.5:1136-1142; pMC1 neo Poly-A, see Thomas, et al. (1987) Cell 51:503-512;and a baculovirus vector such as pAC 373 or pAC 610.

It is likely that CCL28 or CCL27 chemokines need not be glycosylated toelicit biological responses. However, it will occasionally be desirableto express a CCL28 or CCL27 chemokine polypeptide in a system whichprovides a specific or defined glycosylation pattern. In this case, theusual pattern will be that provided naturally by the expression system.However, the pattern will be modifiable by exposing the polypeptide,e.g., in unglycosylated form, to appropriate glycosylating proteinsintroduced into a heterologous expression system. For example, the CCL27chemokine gene may be co-transformed with one or more genes encodingmammalian or other glycosylating enzymes. It is further understood thatover glycosylation may be detrimental to CCL27 chemokine biologicalactivity, and that one of skill may perform routine testing to optimizethe degree of glycosylation which confers optimal biological activity.

A CCL28 or CCL27 chemokine, or a fragment thereof, may be engineered tobe phosphatidyl inositol (PI) linked to a cell membrane, but can beremoved from membranes by treatment with a phosphatidyl inositolcleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. Thisreleases the antigen in a biologically active form, and allowspurification by standard procedures of protein chemistry. See, e.g., Low(1989) Biochem. Biophys. Acta 988:427-454; Tse, et al. (1985) Science230:1003-1008; and Brunner, et al. (1991) J. Cell Biol. 114:1275-1283.

Now that CCL27 and CCL28 chemokines have been characterized, fragmentsor derivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis Springer-Verlag, New York, N.Y.; and Bodanszky (1984)The Principles of Peptide Synthesis Springer-Verlag, New York, N.Y. Forexample, an azide process, an acid chloride process, an acid anhydrideprocess, a mixed anhydride process, an active ester process (forexample, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethylester), a carbodiimidazole process, an oxidative-reductive process, or adicyclohexylcarbodiimide (DCCD)/additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses. See also chemical ligation, e.g., Dawson, et al. (1994)Science 266:776-779, a method of linking long synthetic peptides by apeptide bond.

The prepared protein and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, for example,by extraction, precipitation, electrophoresis and various forms ofchromatography, and the like. The CCL28 or CCL27 chemokines of thisinvention can be obtained in varying degrees of purity depending uponits desired use. Purification can be accomplished by use of knownprotein purification techniques or by the use of the antibodies orbinding partners herein described, e.g., in immunoabsorbant affinitychromatography. This immunoabsorbant affinity chromatography is carriedout by first linking the antibodies to a solid support and thencontacting the linked antibodies with solubilized lysates of appropriatesource cells, lysates of other cells expressing the ligand, or lysatesor supernatants of cells producing the CCL28 or CCL27 chemokines as aresult of recombinant DNA techniques, see below.

Multiple cell lines may be screened for one which expresses a CCL27chemokine at a high level compared with other cells. Natural CCL28 orCCL27 chemokines can be isolated from natural sources, or by expressionfrom a transformed cell using an appropriate expression vector.Purification of the expressed protein is achieved by standardprocedures, or may be combined with engineered means for effectivepurification at high efficiency from cell lysates or supernatants.Epitope or other tags, e.g., FLAG or His₆ segments, can be used for suchpurification features.

V. Antibodies

Antibodies can be raised to various CCL28 or CCL27 chemokines, includingindividual, polymorphic, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in their recombinant forms. Additionally, antibodies can be raisedto CCL28 or CCL27 chemokines in either their active forms or in theirinactive forms. Anti-idiotypic antibodies may also be used. Theantibodies may exhibit various binding specificities for species,individual or polymorphic variants

A. Antibody Production

A number of immunogens may be used to produce antibodies specificallyreactive with CCL28 or CCL27 chemokine proteins. Recombinant protein isthe preferred immunogen for the production of monoclonal or polyclonalantibodies. Naturally occurring protein may also be used either in pureor impure form. Synthetic peptides, made using the CCL28 or CCL27chemokine protein sequences described herein, may also used as animmunogen for the production of antibodies to CCL28 or CCL27 chemokines.Recombinant protein can be expressed in eukaryotic or prokaryotic cellsas described herein, and purified as described. Naturally folded ordenatured material can be used, as appropriate, for producingantibodies. Either monoclonal or polyclonal antibodies may be generatedfor subsequent use in immunoassays to measure the protein.

Methods of producing polyclonal antibodies are known to those of skillin the art. Typically, an immunogen, preferably a purified protein, ismixed with an adjuvant and animals are immunized with the mixture. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to the CCL28or CCL27 chemokine protein of interest. When appropriately high titersof antibody to the immunogen are obtained, usually after repeatedimmunizations, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be done if desired. See, e.g., Harlow andLane; or Coligan.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein (1976) Eur. J. Immunol.6:511-519, incorporated herein by reference). Alternative methods ofimmortalization include transformation with Epstein Barr Virus,oncogenes, or retroviruses, or other methods known in the art. Coloniesarising from single immortalized cells are screened for production ofantibodies of the desired specificity and affinity for the antigen, andyield of the monoclonal antibodies produced by such cells may beenhanced by various techniques, including injection into the peritonealcavity of a vertebrate host. Alternatively, one may isolate DNAsequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according, e.g.,to the general protocol outlined by Huse, et al. (1989) Science246:1275-1281.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of CCL27 chemokines can be raised byimmunization of animals with conjugates of the fragments with carrierproteins as described above. Monoclonal antibodies are prepared fromcells secreting the desired antibody. These antibodies can be screenedfor binding to normal or defective CCL28 or CCL27 chemokines, orscreened for agonistic or antagonistic activity, e.g., mediated througha receptor. These monoclonal antibodies will usually bind with at leasta K_(D) of about 1 mM, more usually at least about 300 μM, typically atleast about 10 μM, more typically at least about 30 μM, preferably atleast about 10 μM, and more preferably at least about 3 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory ManualCSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York, N.Y.; and particularly in Kohler andMilstein (1975) Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed and cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse, et al. (1989) “Generationof a Large Combinatorial Library of the Immunoglobulin Repertoire inPhage Lambda,” Science 246:1275-1281; and Ward, et al. (1989) Nature341:544-546. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen, et al. (1989) Proc. Nat'lAcad. Sci. USA 86:10029-10033.

The antibodies of this invention are useful for affinity chromatographyin isolating CCL28 or CCL27 chemokine protein. Columns can be preparedwhere the antibodies are linked to a solid support, e.g., particles,such as agarose, SEPHADEX, or the like, where a cell lysate orsupernatant may be passed through the column, the column washed,followed by increasing concentrations of a mild denaturant, wherebypurified CCL28 or CCL27 chemokine protein will be released. Likewise,antibody binding to the chemokine may be capable of neutralizingreceptor binding, and may serve as a receptor antagonist. They may alsobe useful as Western blot detection reagents, or ELISA reagents.

The antibodies may also be used to screen expression libraries forparticular expression products. Usually the antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies to CCL28 or CCL27 chemokines may be used for theidentification of cell populations expressing CCL28 or CCL27 chemokines.By assaying the expression products of cells expressing, e.g., CCL27chemokines it is possible to diagnose disease, e.g., immune-compromisedconditions.

Antibodies raised against each CCL28 or CCL27 chemokine will also beuseful to raise anti-idiotypic antibodies. These will be useful indetecting or diagnosing various immunological conditions related toexpression of the antigens.

B. Immunoassays

A particular protein can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Stites and Terr (eds.) (1991) Basic and Clinical Immunology(7th ed.). Moreover, the immunoassays of the present invention can beperformed in many configurations, which are reviewed extensively inMaggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.;Tijan (1985) “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers B. V., Amsterdam; and Harlow and Lane Antibodies, ALaboratory Manual, supra, each of which is incorporated herein byreference. See also Chan (ed.) (1987) Immunoassay: A Practical GuideAcademic Press, Orlando, Fla.; Price and Newman (eds.) (1991) Principlesand Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988)Non-isotopic Immunoassays Plenum Press, NY.

Immunoassays for measurement of, e.g., CCL28 chemokine proteins can beperformed by a variety of methods known to those skilled in the art. Inbrief, immunoassays to measure the protein can be either competitive ornoncompetitive binding assays. In competitive binding assays, the sampleto be analyzed competes with a labeled analyte for specific bindingsites on a capture agent bound to a solid surface. Preferably thecapture agent is an antibody specifically reactive with CCL28 chemokineproteins produced as described above. The concentration of labeledanalyte bound to the capture agent is inversely proportional to theamount of free analyte present in the sample.

In a competitive binding immunoassay, the CCL28 chemokine proteinpresent in the sample competes with labeled protein for binding to aspecific binding agent, for example, an antibody specifically reactivewith the CCL28 chemokine protein. The binding agent may be bound to asolid surface to effect separation of bound labeled protein from theunbound labeled protein. Alternately, the competitive binding assay maybe conducted in liquid phase and a variety of techniques known in theart may be used to separate the bound labeled protein from the unboundlabeled protein. Following separation, the amount of bound labeledprotein is determined. The amount of protein present in the sample isinversely proportional to the amount of labeled protein binding.

Alternatively, a homogeneous immunoassay may be performed in which aseparation step is not needed. In these immunoassays, the label on theprotein is altered by the binding of the protein to its specific bindingagent. This alteration in the labeled protein results in a decrease orincrease in the signal emitted by label, so that measurement of thelabel at the end of the immunoassay allows for detection or quantitationof the protein.

CCL28 or CCL27 chemokine proteins may also be determined by a variety ofnoncompetitive immunoassay methods. For example, a two-site, solid phasesandwich immunoassay may be used. In this type of assay, a binding agentfor the protein, for example an antibody, is attached to a solidsupport. A second protein binding agent, which may also be an antibody,and which binds the protein at a different site, is labeled. Afterbinding at both sites on the protein has occurred, the unbound labeledbinding agent is removed and the amount of labeled binding agent boundto the solid phase is measured. The amount of labeled binding agentbound is directly proportional to the amount of protein in the sample.

Western blot analysis can be used to determine the presence of, e.g.,CCL27 chemokine proteins in a sample. Electrophoresis is carried out,for example, on a tissue sample suspected of containing the protein.Following electrophoresis to separate the proteins, and transfer of theproteins to a suitable solid support, e.g., a nitrocellulose filter, thesolid support is incubated with an antibody reactive with the protein.This antibody may be labeled, or alternatively may be detected bysubsequent incubation with a second labeled antibody that binds theprimary antibody.

The immunoassay formats described above employ labeled assay components.The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. A wide varietyof labels and methods may be used. Traditionally, a radioactive labelincorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P was used. Non-radioactivelabels include ligands which bind to labeled antibodies, fluorophores,chemiluminescent agents, enzymes, and antibodies which can serve asspecific binding pair members for a labeled ligand. The choice of labeldepends on sensitivity required, ease of conjugation with the compound,stability requirements, and available instrumentation. For a review ofvarious labeling or signal producing systems which may be used, see U.S.Pat. No. 4,391,904, which is incorporated herein by reference.

Antibodies reactive with a particular protein can also be measured by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures applicable to the measurement of antibodies byimmunoassay techniques, see Stites and Terr (eds.) Basic and ClinicalImmunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra; andHarlow and Lane Antibodies, A Laboratory Manual, supra.

In brief, immunoassays to measure antisera reactive with, e.g., CCL28chemokine proteins can be either competitive or noncompetitive bindingassays. In competitive binding assays, the sample analyte competes witha labeled analyte for specific binding sites on a capture agent bound toa solid surface. Preferably the capture agent is a purified recombinantCCL28 chemokine protein produced as described above. Other sources ofCCL28 chemokine proteins, including isolated or partially purifiednaturally occurring protein, may also be used. Noncompetitive assaysinclude sandwich assays, in which the sample analyte is bound betweentwo analyte-specific binding reagents. One of the binding agents is usedas a capture agent and is bound to a solid surface. The second bindingagent is labeled and is used to measure or detect the resultant complexby visual or instrument means. A number of combinations of capture agentand labeled binding agent can be used. A variety of differentimmunoassay formats, separation techniques, and labels can be also beused similar to those described above for the measurement of CCL27chemokine proteins.

VI. Purified CCL28 or CCL27 Chemokines

Human CCL28 nucleotide and amino acid sequence is provided in SEQ ID NO:1 and 2. Human CCL27 nucleotide and amino acid sequence is provided inSEQ ID NO: 5 and 6; mouse CCL27 nucleotide and amino acid sequence isprovided in SEQ ID NO: 7 and 8.

Purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatepolyclonal and monoclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene, N.Y.; and Harlow and Lane (1989)Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, which areincorporated herein by reference. Alternatively, a CCL28 or CCL27chemokine receptor can be useful as a specific binding reagent, andadvantage can be taken of its specificity of binding, for, e.g.,purification of a CCL27 chemokine ligand.

The specific binding composition can be used for screening an expressionlibrary made from a cell line which expresses a CCL27 chemokine. Manymethods for screening are available, e.g., standard staining of surfaceexpressed ligand, or by panning. Screening of intracellular expressioncan also be performed by various staining or immunofluorescenceprocedures. The binding compositions could be used to affinity purify orsort out cells expressing the ligand.

The peptide segments, along with comparison to homologous genes, canalso be used to produce appropriate oligonucleotides to screen alibrary. The genetic code can be used to select appropriateoligonucleotides useful as probes for screening. In combination withpolymerase chain reaction (PCR) techniques, synthetic oligonucleotideswill be useful in selecting desired clones from a library, includingnatural allelic an polymorphic variants.

The peptide sequences allow preparation of peptides to generateantibodies to recognize such segments, and allow preparation ofoligonucleotides which encode such sequences. The sequence also allowsfor synthetic preparation, e.g., see Dawson, et al. (1994) Science266:776-779. Since CCL27 and CCL28 chemokines may be secreted proteins,the gene will normally possess an N-terminal signal sequence, which isremoved upon processing and secretion. However, the exact processingpoint may be vary in different cell types, and forms of differentlengths are often detected. Prediction of the signal cleavage point canbe performed, e.g., using the methods of Nielsen, et al. (1997) ProteinEng. 10:1-8. Analysis of the structural features in comparison with themost closely related reported sequences has revealed similarities withother cytokines, particularly the class of proteins known as CC and CXCchemokines.

VII. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence similarity with an amino acid sequence of a CCL28 orCCL27 chemokine. Natural variants include individual, polymorphic,allelic, strain, or species variants.

Amino acid sequence similarity, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. This changes when considering conservative substitutions asmatches. Conservative substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Homologousamino acid sequences include natural polymorphic, allelic, andinterspecies variations in the protein sequence. Typical homologousproteins or peptides will have from 50-100% similarity (if gaps can beintroduced), to 75-100% similarity (if conservative substitutions areincluded) with the amino acid sequence of the CCL27 chemokine.Similarity measures will be at least about 50%, generally at least 60%,more generally at least 65%, usually at least 70%, more usually at least75%, preferably at least 80%, and more preferably at least 80%, and inparticularly preferred embodiments, at least 85% or more. See alsoNeedleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al.(1983) Time Warps, String Edits, and Macromolecules: The Theory andPractice of Sequence Comparison Chapter One, Addison-Wesley, Reading,Mass.; and software packages from IntelliGenetics, Mountain View,Calif.; and the University of Wisconsin Genetics Computer Group,Madison, Wis.

Nucleic acids encoding mammalian CCL28 chemokine proteins will typicallyhybridize to the nucleic acid sequence of SEQ ID NO: 1 under stringentconditions. For example, nucleic acids encoding CCL28 chemokine proteinswill normally hybridize to the nucleic acid of SEQ ID NO: 1 understringent hybridization conditions. Generally, stringent conditions areselected to be about 10° C. lower than the thermal melting point (Tm)for the probe sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration isabout 0.2 molar at pH 7 and the temperature is at least about 50° C.Other factors may significantly affect the stringency of hybridization,including, among others, base composition and size of the complementarystrands, the presence of organic solvents such as formamide, and theextent of base mismatching. A preferred embodiment will include nucleicacids which will bind to disclosed sequences in 50% formamide and 200 mMNaCl at 42° C.

An isolated CCL27 chemokine DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, and shortinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode CCL28 or CCL27 chemokine antigens, theirderivatives, or proteins having highly similar physiological,immunogenic, or antigenic activity.

Modified sequences can be used to produce mutant antigens or to enhanceexpression. Enhanced expression may involve gene amplification,increased transcription, increased translation, and other mechanisms.Such mutant CCL27 chemokine derivatives include predetermined orsite-specific mutations of the protein or its fragments. “Mutant CCL27chemokine” encompasses a polypeptide otherwise falling within thehomology definition of the human CCL27 chemokine as set forth above, buthaving an amino acid sequence which differs from that of a CCL27chemokine as found in nature, whether by way of deletion, substitution,or insertion. In particular, “site specific mutant CCL27 chemokine”generally includes proteins having significant similarity with a proteinhaving a sequence of SEQ ID NO: 6 or 8, e.g., natural embodiments, andas sharing various biological activities, e.g., antigenic orimmunogenic, with those sequences, and in preferred embodiments containmost or all of the disclosed sequence. This applies also to polymorphicvariants from different individuals. Similar concepts apply to differentCCL28 or CCL27 chemokine proteins, particularly those found in variouswarm blooded animals, e.g., mammals and birds. As stated before, it isemphasized that descriptions are generally meant to encompass otherCCL28 or CCL27 chemokine proteins, not limited to the mouse or humanembodiments specifically discussed.

Although site specific mutation sites are predetermined, mutants neednot be site specific. For example, CCL27 chemokine mutagenesis can beconducted by making amino acid insertions or deletions. Substitutions,deletions, insertions, or any combinations may be generated to arrive ata final construct. Insertions include amino- or carboxyl-terminalfusions, e.g. epitope tags. Random mutagenesis can be conducted at atarget codon and the expressed mutants can then be screened for thedesired activity. Methods for making substitution mutations atpredetermined sites in DNA having a known sequence are well known in theart, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR)techniques. See also, Sambrook, et al. (1989) and Ausubel, et al. (1987and Supplements). The mutations in the DNA normally should not placecoding sequences out of reading frames and preferably will not createcomplementary regions that could hybridize to produce secondary mRNAstructure such as loops or hairpins.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of an immunoglobulin with a CCL27 chemokine polypeptide is acontinuous protein molecule having sequences fused in a typical peptidelinkage, typically made as a single translation product and exhibitingproperties derived from each source peptide. A similar concept appliesto heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, protein-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.Thus, new chimeric polypeptides exhibiting new combinations ofspecificities will result from the functional linkage of protein-bindingspecificities and other functional domains.

VIII. Binding Agent:chemokine Protein Complexes

A CCL28 or CCL27 chemokine protein that specifically, or selectively,binds to or that is specifically immunoreactive with an antibodygenerated against a defined immunogen, such as an immunogen consistingof the amino acid sequence of SEQ ID NO: 2 or 8, is typically determinedin an immunoassay. The immunoassay uses a polyclonal antiserum which wasraised to a protein of SEQ ID NO: 2, 6, or 8. This antiserum is selectedto have low crossreactivity against other chemokines and any suchcrossreactivity is removed by immunoabsorption prior to use in theimmunoassay.

In order to produce antisera for use in an immunoassay, the protein ofSEQ ID NO: 2 or 6 or 8, is isolated as described herein. For example,recombinant protein may be produced in a mammalian cell line. An inbredstrain of mice such as BALB/c is immunized with the protein of SEQ IDNO: 2 or 6 or 8, using a standard adjuvant, such as Freund's adjuvant,and a standard mouse immunization protocol (see Harlow and Lane, supra).Alternatively, a synthetic peptide, preferably near full length, derivedfrom the sequences disclosed herein and conjugated to a carrier proteincan be used an immunogen. Polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Polyclonal antisera with a titer of 10₄ or greater are selected andtested for their cross reactivity against C, CC, CX3C, and CXCchemokines, using a competitive binding immunoassay such as the onedescribed in Harlow and Lane, supra, at pages 570-573. Preferably twochemokines are used in this determination in conjunction with humanCCL27 chemokine.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, a protein of SEQ ID NO: 2or of SEQ ID NO: 6 and/or 8 can be immobilized to a solid support.Proteins added to the assay compete with the binding of the antisera tothe immobilized antigen. The ability of the above proteins to competewith the binding of the antisera to the immobilized protein is comparedto the protein of SEQ ID NO: 2 or of SEQ ID NO: 6 and/or 8. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the proteins listed above are selected and pooled. Thecross-reacting antibodies are then removed from the pooled antisera byimmunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (e.g., the CCL27 chemokine motif of SEQ ID NO: 6or 8). In order to make this comparison, the two proteins are eachassayed at a wide range of concentrations and the amount of each proteinrequired to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired is less than twice the amount of the protein, e.g., of SEQ IDNO: 6 or 8 that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

It is understood that CCL27 chemokine protein is a species form of agroup of homologous proteins across species that include closely relatedgenes. For a particular gene product, such as the CCL27 chemokineprotein, the term refers not only to the amino acid sequences disclosedherein, but also to other proteins that are polymorphic, allelic, ornon-allelic variants. It is also understood that the term “CCL27”includes nonnatural mutations introduced by deliberate mutation usingconventional recombinant technology such as single site mutation, or byexcising short sections of DNA encoding CCL27 chemokine proteins, or bysubstituting new amino acids, or adding new amino acids. Such minoralterations should substantially maintain the immunoidentity of theoriginal molecule and/or its biological activity. Thus, thesealterations include proteins that are specifically immunoreactive with adesignated naturally occurring CCL27 chemokine protein, for example, theCCL27 chemokine protein shown in SEQ ID NO: 6 or 8. The biologicalproperties of the altered proteins can be determined by expressing theprotein in an appropriate cell line and measuring an appropriatebiological activity, e.g., a chemotactic effect. Particular proteinmodifications considered minor would include conservative substitutionof amino acids with similar chemical properties, as described above forthe CCL27 chemokine as a whole. By aligning a protein optimally with theprotein of SEQ ID NO: 6 or 8, and by using the conventional immunoassaysdescribed herein to determine immunoidentity, or by using lymphocytechemotaxis assays, one can determine the protein compositions of theinvention.

IX. Functional Variants

The blocking of physiological response to CCL28 or CCL27 chemokine mayresult from the inhibition of binding of the protein to its receptor,e.g., through competitive inhibition. Thus, in vitro assays of thepresent invention will often use isolated protein, membranes from cellsexpressing a recombinant membrane associated CCL28 or CCL27 chemokine,soluble fragments comprising receptor binding segments of theseproteins, or fragments attached to solid phase substrates. These assayswill also allow for the diagnostic determination of the effects ofeither binding segment mutations and modifications, or protein mutationsand modifications, e.g., protein analogs. This invention alsocontemplates the use of competitive drug screening assays, e.g., whereneutralizing antibodies to antigen or receptor fragments compete with atest compound for binding to the protein. In this manner, the antibodiescan be used to detect the presence of a polypeptide which shares one ormore antigenic binding sites of the protein and can also be used tooccupy binding sites on the protein that might otherwise interact with areceptor.

“Derivatives” of, e.g., CCL27 chemokine antigens include amino acidsequence mutants, glycosylation variants, and covalent or aggregateconjugates with other chemical moieties. Covalent derivatives can beprepared by linkage of functionalities to groups which are found inCCL27 chemokine amino acid side chains or at the N- or C-termini, bymeans which are well known in the art. These derivatives can include,without limitation, aliphatic esters or amides of the carboxyl terminus,or of residues containing carboxyl side chains, O-acyl derivatives ofhydroxyl group-containing residues, and N-acyl derivatives of the aminoterminal amino acid or amino-group containing residues, e.g., lysine orarginine. Acyl groups are selected from the group of alkyl-moietiesincluding, e.g., C3 to C18 normal alkyl, thereby forming alkanoyl aroylspecies. Covalent attachment to carrier proteins may be important whenimmunogenic moieties are haptens.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine, or other moieties, including ribosylgroups or cross-linking reagents.

A major group of derivatives are covalent conjugates of the CCL27chemokine or fragments thereof with other proteins or polypeptides.These derivatives can be synthesized in recombinant culture such as N-or C-terminal fusions or by the use of agents known in the art for theirusefulness in cross-linking proteins through reactive side groups.Preferred protein derivatization sites with cross-linking agents are atfree amino groups, carbohydrate moieties, and cysteine residues.

Fusion polypeptides between CCL27 chemokine and other homologous orheterologous proteins are also provided. Many growth factors andcytokines are homodimeric entities, and a repeat construct may havevarious advantages, including lessened susceptibility to proteolyticdegradation. Moreover, many receptors require ligand dimerization totransduce a signal, and various dimeric proteins or domain repeats canbe desirable. Heterologous polypeptides may be fusions between differentsurface markers, resulting in, e.g., a hybrid protein exhibitingreceptor binding specificity. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of aprotein, e.g., a receptor-binding segment, so that the presence orlocation of the fused protein may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609. Other gene fusion partners includebacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase,alcohol dehydrogenase, and yeast alpha mating factor. See, e.g.,Godowski, et al. (1988) Science 241:812-816.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those which havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity ligands.

This invention also contemplates the use of derivatives of CCL27chemokine other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into the threeclasses: (1) salts, (2) side chain and terminal residue covalentmodifications, and (3) adsorption complexes, for example with cellmembranes. Such covalent or aggregative derivatives are useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of ligands or other binding ligands. Forexample, a CCL27 chemokine antigen can be immobilized by covalentbonding to a solid support such as cyanogen bromide-activated SEPHAROSE,by methods which are well known in the art, or adsorbed onto polyolefinsurfaces, with or without glutaraldehyde cross-linking, for use in theassay or purification of anti-CCL27 chemokine antibodies or itsreceptor. The CCL27 chemokine can also be labeled with a detectablegroup, e.g., radioiodinated by the chloramine T procedure, covalentlybound to rare earth chelates, or conjugated to another fluorescentmoiety for use in diagnostic assays. Purification of CCL27 chemokinesmay be effected by immobilized antibodies or receptor.

Isolated CCL27 chemokine genes will allow transformation of cellslacking expression of corresponding CCL27 chemokine, e.g., eitherspecies types or cells which lack corresponding proteins and exhibitnegative background activity. Expression of transformed genes will allowisolation of antigenically pure cell lines, with defined or singlespecie variants. This approach will allow for more sensitive detectionand discrimination of the physiological effects of CCL27 chemokinereceptor proteins. Subcellular fragments, e.g., cytoplasts or membranefragments, can be isolated and used. Descriptions using CCL27 as anexample will generally be alternatively applicable to the CCL28.

X. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for developmental abnormalities, or below in thedescription of kits for diagnosis.

CCL27 chemokine nucleotides, e.g., CCL27 chemokine DNA or RNA, may beused as a component in a forensic assay. For instance, the nucleotidesequences provided may be labeled using, e.g., ³²P or biotin and used toprobe standard restriction fragment polymorphism blots, providing ameasurable character to aid in distinguishing between individuals or,e.g., species sources. Such probes may be used in well-known forensictechniques such as genetic fingerprinting. In addition, nucleotideprobes made from CCL27 chemokine sequences may be used in situ assays todetect chromosomal abnormalities. For instance, rearrangements in thehuman chromosome encoding a CCL27 chemokine gene may be detected viawell-known in situ techniques, using CCL27 chemokine probes inconjunction with other known chromosome markers.

Antibodies and other binding agents directed towards CCL27 chemokineproteins or nucleic acids may be used to purify the corresponding CCL27chemokine molecule. As described in the Examples below, antibodypurification of CCL27 chemokine components is both possible andpracticable. Antibodies and other binding agents may also be used in adiagnostic fashion to determine whether CCL27 chemokine components arepresent in a tissue sample or cell population using well-knowntechniques described herein. The ability to attach a binding agent to aCCL27 chemokine provides a means to diagnose disorders associated withCCL27 chemokine misregulation. Antibodies and other CCL27 chemokinebinding agents may also be useful as histological markers. As describedin the examples below, CCL27 chemokine expression is limited to specifictissue types. By directing a probe, such as an antibody or nucleic acidto a CCL27 chemokine it is possible to use the probe to distinguishtissue and cell types in situ or in vitro.

This invention also provides reagents with significant therapeuticvalue. The CCL27 chemokine (naturally occurring or recombinant),fragments thereof, and antibodies thereto, along with compoundsidentified as having binding affinity to a CCL27 chemokine, are usefulin the treatment of conditions associated with abnormal physiology ordevelopment, including abnormal proliferation, e.g., cancerousconditions, or degenerative conditions. Abnormal proliferation,regeneration, degeneration, and atrophy may be modulated by appropriatetherapeutic treatment using the compositions provided herein. Forexample, a disease or disorder associated with abnormal expression orabnormal signaling by a CCL27 chemokine is a target for an agonist orantagonist of the protein. The proteins likely play a role in regulationor development of neuronal or hematopoietic cells, e.g., lymphoid cells,which affect immunological responses.

Other abnormal developmental conditions are known in cell types shown topossess CCL28 or CCL27 chemokine mRNA by northern blot analysis. SeeBerkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co.,Rahway, N.J.; and Thorn, et al. Harrison's Principles of InternalMedicine, McGraw-Hill, N.Y. Developmental or functional abnormalities,e.g., of the neuronal or immune system, cause significant medicalabnormalities and conditions which may be susceptible to prevention ortreatment using compositions provided herein.

Certain chemokines have also been implicated in viral replicationmechanisms. See, e.g., Cohen (1996) Science 272:809-810; Feng, et al.(1996) Science 272:872-877; and Cocchi, et al. (1995) Science270:1811-1816. The CCL28 or CCL27 chemokine may be useful in a similarcontext.

Recombinant CCL28 or CCL27 chemokine or chemokine antibodies can bepurified and then administered to a patient. These reagents can becombined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers and excipients. These combinations can be sterilefiltered and placed into dosage forms as by lyophilization in dosagevials or storage in stabilized aqueous preparations. This invention alsocontemplates use of antibodies or binding fragments thereof, includingforms which are not complement binding.

Drug screening using antibodies or receptor or fragments thereof canidentify compounds having binding affinity to CCL28 or CCL27 chemokine,including isolation of associated components. Subsequent biologicalassays can then be utilized to determine if the compound has intrinsicstimulating activity and is therefore a blocker or antagonist in that itblocks the activity of the protein. Likewise, a compound havingintrinsic stimulating activity can activate the receptor and is thus anagonist in that it simulates the activity of, e.g., a CCL27 chemokine.This invention further contemplates the therapeutic use of antibodies toCCL27 chemokine as antagonists. This approach should be particularlyuseful with other CCL27 chemokine species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics(8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous administration.

CCL28 or CCL27 chemokines, fragments thereof, and antibodies to it orits fragments, antagonists, and agonists, may be administered directlyto the host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in any conventional dosage formulation. While it ispossible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8thed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: TabletsDekker, NY; and Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Disperse Systems Dekker, NY. The therapy of this invention may becombined with or used in association with other therapeutic agents.

Both the naturally occurring and the recombinant forms of the CCL28 orCCL27 chemokines of this invention are particularly useful in kits andassay methods which are capable of screening compounds for bindingactivity to the proteins. Several methods of automating assays have beendeveloped in recent years so as to permit screening of tens of thousandsof compounds in a short period. See, e.g., Fodor, et al. (1991) Science251:767-773, and other descriptions of chemical diversity libraries,which describe means for testing of binding affinity by a plurality ofcompounds. The development of suitable assays can be greatly facilitatedby the availability of large amounts of purified, soluble CCL27chemokine as provided by this invention.

For example, antagonists can normally be found once the protein has beenstructurally defined. Testing of potential protein analogs is nowpossible upon the development of highly automated assay methods using apurified receptor. In particular, new agonists and antagonists will bediscovered by using screening techniques described herein. Of particularimportance are compounds found to have a combined binding affinity formultiple CCL27 chemokine receptors, e.g., compounds which can serve asantagonists for species variants of a CCL27 chemokine.

This invention is particularly useful for screening compounds by usingrecombinant protein in a variety of drug screening techniques. Theadvantages of using a recombinant protein in screening for specificligands include: (a) improved renewable source of the CCL27 chemokinefrom a specific source; (b) potentially greater number of ligands percell giving better signal to noise ratio in assays; and (c) speciesvariant specificity (theoretically giving greater biological and diseasespecificity).

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing a chemokine receptor. Cells may be isolated which express areceptor in isolation from any others. Such cells, either in viable orfixed form, can be used for standard ligand/receptor binding assays. Seealso, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells (source of CCL27 chemokine) arecontacted and incubated with a labeled receptor or antibody having knownbinding affinity to the ligand, such as ¹²⁵I-antibody, and a test samplewhose binding affinity to the binding composition is being measured. Thebound and free labeled binding compositions are then separated to assessthe degree of ligand binding. The amount of test compound bound isinversely proportional to the amount of labeled receptor binding to theknown source. Any one of numerous techniques can be used to separatebound from free ligand to assess the degree of ligand binding. Thisseparation step could typically involve a procedure such as adhesion tofilters followed by washing, adhesion to plastic followed by washing, orcentrifugation of the cell membranes. Viable cells could also be used toscreen for the effects of drugs on CCL27 chemokine mediated functions,e.g., second messenger levels, i.e., Ca⁺⁺; cell proliferation; inositolphosphate pool changes; and others. Some detection methods allow forelimination of a separation step, e.g., a proximity sensitive detectionsystem. Calcium sensitive dyes will be useful for detecting Ca⁺⁺ levels,with a fluorimeter or a fluorescence cell sorting apparatus.

Another method utilizes membranes from transformed eukaryotic orprokaryotic host cells as the source of a CCL27 chemokine. These cellsare stably transformed with DNA vectors directing the expression of aCCL27 chemokine, e.g., an engineered membrane bound form. Essentially,the membranes would be prepared from the cells and used in areceptor/ligand binding assay such as the competitive assay set forthabove.

Still another approach is to use solubilized, unpurified or solubilized,purified CCL27 chemokine from transformed eukaryotic or prokaryotic hostcells. This allows for a “molecular” binding assay with the advantagesof increased specificity, the ability to automate, and high drug testthroughput.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto a CCL27 chemokine antibody and is described in detail in Geysen,European Patent Application 84/03564, published on Sep. 13, 1984. First,large numbers of different small peptide test compounds are synthesizedon a solid substrate, e.g., plastic pins or some other appropriatesurface, see Fodor, et al., supra. Then all the pins are reacted withsolubilized, unpurified or solubilized, purified CCL27 chemokineantibody, and washed. The next step involves detecting bound CCL27chemokine antibody.

Rational drug design may also be based upon structural studies of themolecular shapes of the CCL27 chemokine and other effectors or analogs.See, e.g., Methods in Enzymology vols. 202 and 203. Effectors may beother proteins which mediate other functions in response to ligandbinding, or other proteins which normally interact with the receptor.One means for determining which sites interact with specific otherproteins is a physical structure determination, e.g., x-raycrystallography or 2 dimensional NMR techniques. These will provideguidance as to which amino acid residues form molecular contact regions.For a detailed description of protein structural determination, see,e.g., Blundell and Johnson (1976) Protein Crystallography AcademicPress, NY.

A purified CCL27 chemokine can be coated directly onto plates for use inthe aforementioned drug screening techniques. However, non-neutralizingantibodies to these ligands can be used as capture antibodies toimmobilize the respective ligand on the solid phase. Examples with CCL27will alternately be performed with the CCL28 chemokine.

XI. Kits

This invention also contemplates use of CCL28 or CCL27 chemokineproteins, fragments thereof, peptides, and their fusion products in avariety of diagnostic kits and methods for detecting the presence ofchemokine or a chemokine receptor. Typically the kit will have acompartment containing either a defined CCL28 or CCL27 chemokine peptideor gene segment or a reagent which recognizes one or the other, e.g.,receptor fragments or antibodies.

For example, a kit for determining the binding affinity of a testcompound to a CCL27 chemokine would typically comprise a test compound;a labeled compound, e.g., a receptor or antibody having known bindingaffinity for the CCL27 chemokine; a source of CCL27 chemokine (naturallyoccurring or recombinant); and a means for separating bound from freelabeled compound, such as a solid phase for immobilizing the CCL27chemokine. Once compounds are screened, those having suitable bindingaffinity to the CCL27 chemokine can be evaluated in suitable biologicalassays, as are well known in the art, to determine whether they act asagonists or antagonists to the receptor. The availability of recombinantCCL27 chemokine polypeptides also provide well defined standards forcalibrating such assays.

A preferred kit for determining the concentration of, for example, aCCL27 chemokine in a sample would typically comprise a labeled compound,e.g., receptor or antibody, having known binding affinity for the CCL27chemokine, a source of CCL27 chemokine (naturally occurring orrecombinant), and a means for separating the bound from free labeledcompound, for example, a solid phase for immobilizing the CCL27chemokine. Compartments containing reagents, and instructions, willnormally be provided.

Antibodies, including antigen binding fragments, specific for the CCL27chemokine or ligand fragments are useful in diagnostic applications todetect the presence of elevated levels of CCL27 chemokine and/or itsfragments. Such diagnostic assays can employ lysates, live cells, fixedcells, immunofluorescence, cell cultures, body fluids, and further caninvolve the detection of antigens related to the ligand in serum, or thelike. Diagnostic assays may be homogeneous (without a separation stepbetween free reagent and antigen-CCL27 chemokine complex) orheterogeneous (with a separation step). Various commercial assays exist,such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassaytechnique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), andthe like. For example, unlabeled antibodies can be employed by using asecond antibody which is labeled and which recognizes the antibody to aCCL27 chemokine or to a particular fragment thereof. Similar assays havealso been extensively discussed in the literature. See, e.g., Harlow andLane (1988) Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.)(1987) Immunoassay: A Practical Guide Academic Press, Orlando, Fla.;Price and Newman (eds.) (1991) Principles and Practice of ImmunoassayStockton Press, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay PlenumPress, NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a CCL27 chemokine, as such may be diagnostic ofvarious abnormal states. For example, overproduction of CCL27 chemokinemay result in production of various immunological or other medicalreactions which may be diagnostic of abnormal physiological states,e.g., in cell growth, activation, or differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or receptor, or labeled CCL27chemokine is provided. This is usually in conjunction with otheradditives, such as buffers, stabilizers, materials necessary for signalproduction such as substrates for enzymes, and the like. Preferably, thekit will also contain instructions for proper use and disposal of thecontents after use. Typically the kit has compartments for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification, or may be modifiedin a variety of ways. For example, labeling may be achieved bycovalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In any of these assays, theprotein, test compound, CCL27 chemokine, or antibodies thereto can belabeled either directly or indirectly. Possibilities for direct labelinginclude label groups: radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No.3,645,090) such as peroxidase and alkaline phosphatase, and fluorescentlabels (U.S. Pat. No. 3,940,475) capable of monitoring the change influorescence intensity, wavelength shift, or fluorescence polarization.Possibilities for indirect labeling include biotinylation of oneconstituent followed by binding to avidin coupled to one of the abovelabel groups.

There are also numerous methods of separating the bound from the freeligand, or alternatively the bound from the free test compound. TheCCL27 chemokine can be immobilized on various matrices followed bywashing. Suitable matrices include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the CCL27 chemokine to amatrix include, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin-avidin. The last step inthis approach involves the precipitation of ligand/receptor orligand/antibody complex by any of several methods including thoseutilizing, e.g., an organic solvent such as polyethylene glycol or asalt such as ammonium sulfate. Other suitable separation techniquesinclude, without limitation, the fluorescein antibody magnetizableparticle method described in Rattle, et al. (1984) Clin. Chem.30:1457-1461, and the double antibody magnetic particle separation asdescribed in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of aCCL27 chemokine. These sequences can be used as probes for detectinglevels of the CCL27 chemokine message in samples from natural sources,or patients suspected of having an abnormal condition, e.g., cancer ordevelopmental problem. The preparation of both RNA and DNA nucleotidesequences, the labeling of the sequences, and the preferred size of thesequences has received ample description and discussion in theliterature. Normally an oligonucleotide probe should have at least about14 nucleotides, usually at least about 18 nucleotides, and thepolynucleotide probes may be up to several kilobases. Various labels maybe employed, most commonly radionuclides, particularly ³²P. However,other techniques may also be employed, such as using biotin modifiednucleotides for introduction into a polynucleotide. The biotin thenserves as the site for binding to avidin or antibodies, which may belabeled with a wide variety of labels, such as radionuclides,fluorophores, enzymes, or the like. Alternatively, antibodies may beemployed which can recognize specific duplexes, including DNA duplexes,RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay carried out where theduplex is bound to a surface, so that upon the formation of duplex onthe surface, the presence of antibody bound to the duplex can bedetected. The use of probes to the novel anti-sense RNA may be carriedout using many conventional techniques such as nucleic acidhybridization, plus and minus screening, recombinational probing, hybridreleased translation (HRT), and hybrid arrested translation (HART). Thisalso includes amplification techniques such as polymerase chain reaction(PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of these and other markers are also contemplated. Diagnosis orprognosis may depend on the combination of multiple indications used asmarkers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97.Qualitative or quantitative expression of each chemokine may beevaluated by standard methods at the protein or mRNA levels.

XII. Receptor Isolation

Having isolated a binding partner of a specific interaction, methodsexist for isolating the counter-partner. See, Gearing, et al. (1989)EMBO J. 8:3667-3676. For example, means to label a CCL28 or CCL27chemokine without interfering with the binding to its receptor can bedetermined. For example, an affinity label or epitope tag can be fusedto either the amino- or carboxyl-terminus of the ligand. An expressionlibrary can be screened for specific binding of the CCL28 or CCL27chemokine, e.g., by cell sorting, or other screening to detectsubpopulations which express such a binding component. See, e.g., Ho, etal. (1993) Proc. Nat'l Acad. Sci. USA 90:11267-11271. Alternatively, apanning method may be used. See, e.g., Seed and Aruffo (1987) Proc.Nat'l Acad. Sci. USA 84:3365-3369. A two-hybrid selection system mayalso be applied making appropriate constructs with the availablechemokine sequences. See, e.g., Fields and Song (1989) Nature340:245-246. Standard Ca⁺⁺ flux methods can also be utilized. See, e.g.,Coligan, et al. (eds.) (1992 and periodic supplements) Current Protocolsin Immunology Greene/Wiley, New York, N.Y.

Protein cross-linking techniques with label can be applied to isolatebinding partners of a CCL28 or CCL27 chemokine. This would allowidentification of proteins which specifically interact with a CCL28 orCCL27 chemokine, e.g., in a ligand-receptor like manner. Typically, thechemokine family binds to receptors of the seven transmembrane receptorfamily, and the receptor for the CCL28 or CCL27 chemokine is likely toexhibit a similar structure. Thus, it is likely that the receptor willbe found by expression in a system which is capable of expressing such amembrane protein in a form capable of exhibiting ligand bindingcapability.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the invention tospecific embodiments.

EXAMPLES

I. General Methods

Many of the standard methods below are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning. A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3, CSHPress, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, N.Y.; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, NY. Methods for protein purification include such methods asammonium sulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; and manufacturer's literature on use of protein purificationproducts, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond,Calif. Combination with recombinant techniques allow fusion toappropriate segments (epitope tags), e.g., to a FLAG sequence or anequivalent which can be fused, e.g., via a protease-removable sequence.See, e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990)“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, PlenumPress, NY; and Crowe, et al. (1992) QIAexpress: The High LevelExpression & Protein Purification System QUIAGEN, Inc., Chatsworth,Calif.

Standard immunological techniques are described, e.g., in Coligan (1991)Current Protocols in Immunology Wiley/Greene, N.Y.; and Methods inEnzymology volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150,162, and 163. Assays for neural cell biological activities aredescribed, e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules10, Elsevier; Methods in Neurosciences Academic Press; and NeuromethodsHumana Press, Totowa, N.J. Methodology of developmental systems isdescribed, e.g., in Meisami (ed.) Handbook of Human Growth andDevelopmental Biology CRC Press; and Chrispeels (ed.) MolecularTechniques and Approaches in Developmental Biology Interscience.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Isolation of CCL28 or CCL27 Chemokine Clone

A clone encoding the CCL28 or CCL27 chemokine is isolated from a naturalsource by many different possible methods. Given the sequences providedherein, PCR primers or hybridization probes are selected and/orconstructed to isolate either genomic DNA segments or cDNA reversetranscripts. Appropriate cell sources include listed tissues, e.g., skinor epithelial or wound healing libraries. Genetic and polymorphic orallelic variants are isolated by screening a population of individuals.

PCR based detection is performed by standard methods, preferably usingprimers from opposite ends of the coding sequence, but flanking segmentsmight be selected for specific purposes.

Alternatively, hybridization probes are selected. Particular AT or GCcontents of probes are selected depending upon the expected homology andmismatching expected. Appropriate stringency conditions are selected tobalance an appropriate positive signal to background ratio. Successivewashing steps are used to collect clones of greater homology.

Further clones are isolated using an antibody based selection procedure.Standard expression cloning methods are applied including, e.g., FACSstaining of membrane associated expression product. The antibodies areused to identify clones producing a recognized protein. Alternatively,antibodies are used to purify a CCL28 or CCL27 chemokine, with proteinsequencing and standard means to isolate a gene encoding that protein.

Genomic sequence based methods will also allow for identification ofsequences naturally available, or otherwise, which exhibit homology tothe provided sequences.

III. Isolation of a Primate Counterpart for Chemokine Clone

Similar methods are used as above to isolate an appropriate primatechemokine gene from another primate. Similar source materials are usedto isolate natural genes, including genetic, polymorphic, allelic, orstrain variants. Other species variants are also isolated using similarmethods. Alternatively, gene databases may be searched for theappropriate motifs.

IV. Isolation of a Rodent Chemokine Clone

An appropriate rodent source is selected as above, e.g., rat, hamster,etc. Similar methods are utilized to isolate a species variant, thoughthe level of similarity will typically be lower for rodent chemokine ascompared to a human to other primate sequence.

V. Chromosomal Localization

The cDNA is labeled, e.g., nick-translated with biotin-14 dATP andhybridized in situ at a final concentration of 5 ng/μl to metaphasesfrom two normal animals, preferably males. Fluorescence in situhybridization (FISH) method may be modified from that described byCallen, et al. (1990). Ann. Genet. 33:219-221, in that chromosomes arestained before analysis with both propidium iodide (as counter stain)and DAPI (for chromosome identification). Images of metaphasepreparations are captured by a CCD camera and computer enhanced.Identification of the appropriate labeled chromosomes is determined.Localization to the standard locations for such molecule, or differentlocation may also provide information as to function.

The human CCL28 has been localized to human chromosome 9p13.

VI. Expression; Purification; Characterization

With an appropriate clone from above, the coding sequence is insertedinto an appropriate expression vector. This may be in a vectorspecifically selected for a prokaryote, yeast, insect, or highervertebrate, e.g., mammalian expression system. Standard methods areapplied to produce the gene product, preferably as a soluble secretedmolecule, but will, in certain instances, also be made as anintracellular protein. Intracellular proteins typically require celllysis to recover the protein, and insoluble inclusion bodies are acommon starting material for further purification.

With a clone encoding a CCL28 or CCL27 chemokine, recombinant productionmeans are used, although natural forms may be purified from appropriatesources. The protein product is purified by standard methods of proteinpurification, in certain cases, e.g., coupled with immunoaffinitymethods. Immunoaffinity methods are used either as a purification step,as described above, or as a detection assay to determine the separationproperties of the protein.

Preferably, the protein is secreted into the medium, and the solubleproduct is purified from the medium in a soluble form. Alternatively, asdescribed above, inclusion bodies from prokaryotic expression systemsare a useful source of material. Typically, the insoluble protein issolubilized from the inclusion bodies and refolded using standardmethods. Purification methods are developed as described above.

The product of the purification method described above is characterizedto determine many structural features. Standard physical methods areapplied, e.g., amino acid analysis and protein sequencing. The resultingprotein is subjected to CD spectroscopy and other spectroscopic methods,e.g., NMR, ESR, mass spectroscopy, etc. The product is characterized todetermine its molecular form and size, e.g., using gel chromatographyand similar techniques. Understanding of the chromatographic propertieswill lead to more gentle or efficient purification methods.

Prediction of glycosylation sites may be made, e.g., as reported inHansen, et al. (1995) Biochem. J. 308:801-813.

VII. Preparation of Antibodies Against Chemokines

With DNA for expression, or protein produced, e.g., as above, animalsare immunized to produce antibodies. Polyclonal antiserum is raised, insome cases, using non-purified antigen, though the resulting serum willexhibit higher background levels. Preferably, the antigen is purifiedusing standard protein purification techniques, including, e.g.,affinity chromatography using polyclonal serum indicated above. Presenceof specific antibodies is detected using defined synthetic peptidefragments.

Polyclonal serum is raised against a purified antigen, purified asindicated above, or using, e.g., a plurality of, synthetic peptides. Aseries of overlapping synthetic peptides which encompass all of the fulllength sequence, if presented to an animal, will produce serumrecognizing most linear epitopes on the protein. Such an antiserum isused to affinity purify protein, which is, in turn, used to introduceintact full length protein into another animal to produce anotherantiserum preparation.

Similar techniques are used to generate induce monoclonal antibodies toeither unpurified antigen, or, preferably, purified antigen. Theantiserum or antibodies may recognize native protein, or may recognizedenatured antigen. The preparations may be immunoselected, orimmunopurified, as desired.

Polypeptide fragments corresponding to amino acid residues about 31-41,about 52-63, and about 77-98, of SEQ ID NO:6 (CCL27), have an unusuallyhigh antigenicity, according to Parker antigenicity analysis usingMacVector 7.1® from Accelrys, Inc. (San Diego, Calif.). A Wellingantigenicity plot revealed that regions of unusual antigenicity to befragments comprising amino acids 39-41, about 58-72, and about 90-100 ofSEQ ID NO:6, with analysis using MacVector 7.1®. It is contemplated touse one or more of these antigenic regions, or a fragment of one or moreof these antigenic regions, for antibody production. It is not intendedto limit the invention to the use of the described regions and fragmentsfor the production of antibodies.

Polypeptide fragments corresponding to amino acid residues of unusualantigenicity to be fragments comprising amino acids 28-38, 48-51, 70-84,84-122, and 70-122 of SEQ ID NO:2 (CCL28), according to Parkerantigenicity analysis using MacVector 7.1® from Accelrys, Inc. (SanDiego, Calif.). A Welling antigenicity plot revealed regions of unusualantigenicity to be 10-20, 37-42, 61-68, and 95-122, with analysis byMacVector 7.1®. It is contemplated to use one or more of these antigenicregions, or a fragment of one or more of these antigenic regions, forantibody production. It is not intended to limit the invention to theuse of the described regions and fragments for the production ofantibodies.

Antibodies to CCL28 were prepared against three separate peptides, wherethe peptides were synthesized using the predicted amino acid sequence ofCCL28 and used in immunoblotting studies (WO 99/36540 to Papsidero, etal., pp. 48-51).

VIII. Cellular and Tissue Distribution

Distribution of the protein or gene products are determined, e.g., usingimmunohistochemistry with an antibody reagent, as produced above, or byscreening for nucleic acids encoding the chemokine. Hybridization or PCRmethods are used to detect DNA, cDNA, or message content. Histochemistryallows determination of the specific cell types within a tissue whichexpress higher or lower levels of message or DNA. Antibody techniquesare useful to quantitate protein in a biological sample, including aliquid or tissue sample. Immunoassays are developed to quantitateprotein. Also, FACS analysis may be used to evaluate expression in acell population.

The mouse CCL27 sequence has been detected in fetal mouse, 1 sequencefrom 14.5 days post conception, and 3 sequences from 19.5 days postconception. Three sequences have come from adult mouse, 1 each fromliver, placenta, and skin. Northern analysis shows signal in testes ismuch greater than that in brain, which is much greater than that inlung.

CCL27 occurs in the outermost cell layers of murine skin, correspondingto the epidermis, and only rarely in cells of the dermis, according toimmunohistological analysis (Morales, et al. (1999) Proc. Natl. Acad.Sci. USA 96:14470-14475). In skin from healthy human donors, epidermalkeratinocytes contain detectable CCL27, where the most abundantexpression occurs in keratinocytes in the basal layers of the epidermis,and lower levels in keratinocytes of suprabasal layers, according toimmunohistogical analysis (Homey, et al. (2002) Nature Med. 8:157-165).Other studies of human skin revealed that CCL27 is expressed in skin,but not other tissues, where analysis was by Northern blots (Pan, et al.(2000) J. Immunol. 165:2943-2949). CCL27 is also expressed inmacrophages, dendritic cells, and other cells.

In mouse, CCL27 takes two forms, where one bears a signal peptide, whilethe other form appears to localize in the nucleus (Baird, et al. (1999)J. Biol. Chem. 274:33496-33503).

Total RNA was isolated from a variety of cell types and tissues usingRNEasy® mini kit from Qiagen (Valencia, Calif.) using the manufacturer'ssuggested protocols. RNA was reverse transcribed using oligo dT primers,and Multiscribe® reverse transcriptase (Perkin Elmer Applied Biosystems,Foster City, Calif.), according to the manufacturer's protocols. cDNAwas analyzed for the expression of CCL27, using ubiquitin expression asa standard. cDNA was analyzed for the expression of CCL27 (CCL27) andCCR10 genes using a Perkin-Elmer ABI Prism® 7700 Sequence DetectionSystem (Perkin-Elmer Applied Biosystems, Foster City, Calif.).Amplification of cDNA was by Taqman® PCR assay for real-timequantitative PCR (Perkin-Elmer Applied Biosystems, Foster City, Calif.).Quantitation of expression of target genes was calculated by normalizingthe values relative to the expression of ubiquitin.

Results of Taqman® analysis are presented in Table 1

Human Human cell type CCL27 Epithelial cell keratinocyte untreated (++)Epithelial cell keratinocyte activated TNFα + IL-1 (+++) Keratinocytes,untreated (+) Keratinocytes; TNF-α/IL-1β + IL-10, 18 hours (++)Keratinocytes; TNF-α/IL-1β, 18 hours (+++) Keratinocytes; IL-4, 18 hours(+) Keratinocytes; IFN-γ, 18 hours (+) Mouse Mouse cell or tissue typeCCL27 Fibroblast cell resting L cell line (−) Dendritic cell resting exspleen (+, −) Dendritic cell resting ex bone marrow (+++) Macrophageresting ex bone marrow (+++) Macrophage activated LPS + IFNγ +anti-IL-10R ex bone (++) marrow Mesenteric lymph nodes IL-10 knockouttreated IL-10 (+++) Mesenteric lymph nodes IL-10 knockout treated αIL-12(+)

Distribution of CCL28 and Association with Inflammation (GVHD) andCancer.

CCL28 is expressed in the entire gastrointestinal tract, trachea,placenta, pancreas, thyroid gland, salivary glands, and mammary glands,as determined by a Clontech human multiple tissue expression (MTE®)array RNA blot (BD Clontech, Palo Alto, Calif.) (WO 01/92301 of Hromas,pp. 84-85). Immunohistological analysis revealed CCL28 to occur in humancolon, and induced in the colon of patients who had developed graftversus host disease after allogeneic bone marrow transplantation (WO01/92301 of Hromas, pp. 84-85).

Northern blot analysis of human tissues revealed that CCL28 ispredominantly expressed in prostate, colon, spleen, and to a lesserdegree in peripheral blood leukocytes. Analysis of murine tissuesrevealed that CCL28 is expressed mainly in the testis, and to a lesserdegree in kidney and brain (Wang, et al. (2000) J. Biol. Chem.275:22313-22323).

Taqman® analysis was used for tissues and cell lines with limited levelsof mRNA. Mouse CCL28 was expressed in colon, colon from IL-10 knockoutmouse, lung, rag-1 liver, or normal thymus. Mouse CCL28 was expressed instomach of IL-10 knockout mouse, but not in stomach of normal mouse.Mouse CCL28 was expressed in Th1-type T cells, but not in Th2-type Tcells (FIG. 5 in Wang, et al. (2000) J. Biol. Chem. 275:22313-22323).

Taqman analysis of human samples revealed CCL28 to be expressed inpsoriatic skin and normal skin, where expression was about 20-foldgreater in psoriatic skin. CCL28 was expressed in normal thyroid,Hashimoto's thyroidosis thyroid sample, normal colon, ulcerative colitiscolon, and eosinophils, but not in inflamed tonsil, fetal heart, fetalbrain, fetal liver, or various sources of dendritic cells, B cells, Tcells, or unfractionated white blood cells from blood (PBMCs) (FIG. 6 inWang, et al. (2000) J. Biol. Chem. 275:22313-22323).

A polyclonal antibody against a 20-amino acid peptide (residues 78-98)of murine CCL28 was prepared in rabbits (Zymed Laboratories, Inc., SouthSan Francisco, Calif.). The anti-CCL28 antibody was used forimmunohistological analysis of tissue samples, such as mouse gut.Immunohistological analysis of the intestines revealed CCL28 to bepresent in the epithelial cells (Wang, et al. (2000) J. Biol. Chem.275:22313-22323).

CCL28 expression is high in many normal human breast samples, with lowerexpression in other epithelial-enriched tissues, such as salivary gland,colon, and prostate, with analysis by Northern blots. CCL28 expressionis reduced or eliminated in most human breast tumors, as compared tonormal adjacent tissues. In situ hybridization confirms the abovefinding (Mickanin, et al. (2001) Int. J. Oncol. 18:939-944).

CCL28 levels in human blood serum were measured using antibodies raisedagainst peptides from MACK. Only serum from patients with breast cancerprovided a positive signal, and here the immunoblot method revealed twoprotein bands, at 20-30 kDa and 7-12 kDa. Of 31 blood samples frompatients with breast cancer tested, one specimen showed only the 20-30kDa forms, while all others showed both forms. None of the ten serumspecimens from patients with prostate, ovarian, lung, or colon cancergave a positive signal, and none of the serum samples from seven normalpatients gave a positive signal. (WO 99/36540 of Papsidero, et al., pp.49-51).

IX. Microchemotaxis Assays

The pro-migratory activities of CCL27 chemokine are assessed inmicrochemotaxis assays. See, e.g., Bacon, et al. (1988) Br. J.Pharmacol. 95:966-974. Other trafficking assays are also used. See,e.g., Quidling-Jafrbrink, et al. (1995) Eur. J. Immunol. 25:322-327;Koch, et al. (1994) J. Clinical Investigation 93:921-928; and Antony, etal. (1993) J. Immunol. 151:7216-7223.

Chemokines may also be assayed for activity in hemopoietic assays asdescribed, e.g., by H. Broxmeyer. See Bellido, et al. (1995) J. ClinicalInvestigation 95:2886-2895; and Jilka, et al. (1995) Exptl Hematology23:500-506. They may be assayed for angiogenic activities as described,e.g., by Streiter, et al. (1992) Am. J. Pathol. 141:1279-1284. Or for arole in inflammation. See, e.g., Wakefield, et al. (1996) J. SurgicalRes, 64:26-31.

Chemotaxis in response to CCL27 gradients was measured using human whiteblood cells. The identities of migrating and non-migrating cells weredetermined by labeled antibodies that recognized various membrane-boundproteins. CCL27 stimulated chemotaxis of CD4⁺, CLA⁺ memory T cells, butnot of CD4⁺, CLA⁻ memory T cells. CCL27 was also chemotactic towardCD8⁺, CLA⁺ memory T cells, but not to CD8+, CLA⁻ memory T cells. CCL27was not chemotactic to naïve T cells, B cells, monocytes, or neutrophils(Morales, et al. (1999) Proc. Natl. Acad. Sci. USA 96:14470-14475; WO00/38713).

Injection of CCL27 into the skin attracts lymphocytes, as shown byhistological analysis (FIG. 5f of Homey, et al. (2002) supra). Analysisof mRNA expression in skin tissue by real time PCR revealed that theCCL27 skin injection also resulted in increased levels of mRNA codingfor IL-2, CCR10, and the α-chain of LFA (LFA-1α), indicating increasedrecruitment of T cells to the site of injection (FIG. 5g of Homey, etal. (2002) supra; WO 00/38713).

The correlation between CCL27 response and the presence of CLA isconsistent with the finding that only about 5% of T cells innon-inflamed skin express CLA, while most of the T cells of inflamedskin express CLA (Homey, et al. (2000) Nature Med. 8:157-165).

A study of mouse genomic DNA revealed that the mouse genome contains twodifferent CCL27 genes. One of these genes is deleted in the pit strainof mice, a strain with abnormalities in white blood cell migration andimmune response (Nakano and Gunn (2001) J. Immunol. 166:361-369). Theresults from the pit strain of mice implicate CCL27 as a modulator ofleukocyte migration.

Treatment of unfractionated white blood cells (peripheral bloodmononuclear cells, PBMC) with CCL27 results in increased expression ofCD3 and CD4 (Table I of Baird, et al. (1999) J. Biol. Chem.274:33496-33503). CD3 and CD4 are membrane-bound proteins of T cellsthat play a central role in T cell activation (Abbas, et al. (2000)Cellular and Molecular Immunology, 4^(th) ed., W.B. Saunders Col,Philadelphia, Pa.). CD3 and CD4 are also commonly used as markers for Tcells. Thus, CCL27's effect on T cells includes types of cell activationthat are not directly related to migration.

Note that CD3 and CD4 also occur on certain subpopulations ofmacrophages and NK cells (Hewson, et al. (2001) J. Immunol.166:4835-4842; Arase, et al. (2001) J. Immunol. 166:21-25;Toyama-Sorimachi, et al. (2001) J. Immunol. 166:3771-3779). Hence, theresults of the effect of CCL27 on PBMCs may be due, in part, toactivation of macrophages or NK cells.

Exposing keratinocytes to TNF-α plus IL-1β results in a marked inductionof CCL27 mRNA (FIG. 3 of Morales, et al. (1999) Proc. Natl. Acad. Sci.USA 96:14470-14475). This induction of CCL27 by TNF-α plus IL-1βsuggests a role of CCL27 in skin inflammation, because TNF-α and IL-1βboth increase skin inflammation. The relevance of these two cytokines toskin inflammation was demonstrated by the use of anti-TNF-α (lyer, etal. (2002) Br. J. Dermatol. 146:118-121) and anti-IL-1β (Schon, et al.(2001) Clin. Exp. Immunol. 123:505-510) in the treatment of psoriasis.

Human CCL28 attracts resting CD4⁺ T cells and resting CD8⁺ T cells. Tcells activated with anti-CD3 antibody did not respond to CCL28. HumanCCL28 is was found not to be chemotactic towards T cells bearing the β7polypeptide chain. Maximal chemotaxis of T cells was specifically foundat 100-200 nM CCL28 (Wang, et al. (2000) J. Biol. Chem.275:22313-22323).

Transfectant cells bearing CCR10 are attracted by CCL28, as well as byanother chemokine, CCL27. However, transfectant cells bearing CCR3 areattracted by CCL28, but not by CCL27 (Pan, et al. (2000) supra). CCL28is chemotactic towards T cells, where one subpopulation of T cells thatis attracted is CD45R^(lo)CLA^(hi) T cells (FIG. 2B of Pan, et al.(2000) supra). CCL28 is also chemotactic towards eosinophils (FIG. 2C ofPan, et al. (2000) supra). CCL28 is not chemotactic towards naïve ormemory blood B cells.

Eosinophils, which naturally express CCR3, are attracted by CCL28.Migration of eosinophils towards CCL28 was blocked by a monoclonalantibody towards the receptor, CCR3. Hence, CCL27 and CCL28 differfunctionally in that CCL27 attracts cells bearing CCR10, while CCL28attracts cells bearing CCR10 and cells bearing CCR3 (Pan, et al. (2000)supra).X. Biological Activities, Direct and Indirect

A robust and sensitive assay is selected as described above, e.g., onimmune cells, neuronal cells, or stem cells. Chemokine is added to theassay in increasing doses to see if a dose response is detected. Forexample, in a proliferation assay, cells are plated out in plates.Appropriate culture medium is provided, and chemokine is added to thecells in varying amounts. Growth is monitored over a period of timewhich will detect either a direct effect on the cells, or an indirecteffect of the chemokine.

Alternatively, an activation assay or attraction assay is used. Anappropriate cell type is selected, e.g., hematopoietic cells, myeloid(macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (Tcell, B cell, or NK cells), neural cells (neurons, neuroglia,oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitorcells which differentiate to other cell types, e.g., gut crypt cells andundifferentiated cell types.

Other assays will be those which have been demonstrated with otherchemokines. See, e.g., Schall and Bacon (1994) Current Opinion inImmunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &Immunol. 109:97-109.

XI. Structure Activity Relationship

Information on the criticality of particular residues is determinedusing standard procedures and analysis. Standard mutagenesis analysis isperformed, e.g., by generating many different variants at determinedpositions, e.g., at the positions identified above, and evaluatingbiological activities of the variants. This may be performed to theextent of determining positions which modify activity, or to focus onspecific positions to determine the residues which can be substituted toeither retain, block, or modulate biological activity.

Alternatively, analysis of natural variants can indicate what positionstolerate natural mutations. This may result from populational analysisof variation among individuals, or across strains or species. Samplesfrom selected individuals are analyzed, e.g., by PCR analysis andsequencing. This allows evaluation of population polymorphisms.

XII. Screening for Agonists/Antagonists

Agonists or antagonists are screened for ability to induce or blockbiological activity. The candidate compounds, e.g., sequence variants ofnatural CCL27 chemokine, are assayed for their biological activities.Alternatively, compounds are screened, alone or in combinations, todetermine effects on biological activity.

XIII. Isolation of a Receptor for Chemokine

A CCL28 or CCL27 chemokine can be used as a specific binding reagent toidentify its binding partner, by taking advantage of its specificity ofbinding, much like an antibody would be used. A binding reagent iseither labeled as described above, e.g., fluorescence or otherwise, orimmobilized to a substrate for panning methods. The typical chemokinereceptor is a seven transmembrane receptor.

The binding composition, e.g., chemokine, is used to screen anexpression library made from a cell line which expresses a bindingpartner, i.e. receptor. Standard staining techniques are used to detector sort intracellular or surface expressed receptor, or surfaceexpressing transformed cells are screened by panning. Screening ofintracellular expression is performed by various staining orimmunofluorescence procedures. See also McMahan, et al. (1991) EMBO J.10:2821-2832.

Standard Ca⁺⁺ flux protocols, see, e.g., Coligan, et al. (eds.)(1992 andperiodic supplements) Current Protocols in Immunol. Greene/Wiley, NewYork, N.Y., can be used to identify a receptor for CCL27.

For example, on day 0, precoat 2-chamber permanox slides with 1 ml perchamber of fibronectin, 10 ng/ml in PBS, for 30 min at room temperature.Rinse once with PBS. Then plate COS cells at 2-3×10⁵ cells per chamberin 1.5 ml of growth media. Incubate overnight at 37° C.

On day 1 for each sample, prepare 0.5 ml of a solution of 66 μg/mlDEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum free DME. Foreach set, a positive control is prepared, e.g., of human CCL27 chemokinecDNA at 1 and 1/200 dilution, and a negative mock. Rinse cells withserum free DME. Add the DNA solution and incubate 5 hr at 37° C. Removethe medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and washonce with DME. Add 1.5 ml growth medium and incubate overnight.

On day 2, change the medium. On days 3 or 4, the cells are fixed andstained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80° C. after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin (0.1%) with 32 μl/ml of 1 M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Add chemokine or chemokine/antibodycomplex to cells and incubate for 30 min. Wash cells twice withHBSS/saponin. If appropriate, add first antibody for 30 min. Add secondantibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, andincubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABChorseradish peroxidase solution, and preincubate for 30 min. Use, e.g.,1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 mlHBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solutionand incubate for 30 min. Wash cells twice with HBSS, second wash for 2min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of H₂O₂per 5 ml of glass distilled water. Carefully remove chamber and rinseslide in water. Air dry for a few minutes, then add 1 drop of CrystalMount and a cover slip. Bake for 5 min at 85-90° C.

Evaluate positive staining of pools and progressively subclone toisolation of single genes responsible for the binding.

Alternatively, chemokine reagents are used to affinity purify or sortout cells expressing a receptor. See, e.g., Sambrook, et al. or Ausubel,et al.

Another strategy is to screen for a membrane bound receptor by panning.The receptor cDNA is constructed as described above. The ligand can beimmobilized and used to immobilize expressing cells. Immobilization maybe achieved by use of appropriate antibodies which recognize, e.g., aFLAG sequence of a chemokine fusion construct, or by use of antibodiesraised against the first antibodies. Recursive cycles of selection andamplification lead to enrichment of appropriate clones and eventualisolation of receptor expressing clones.

Phage expression libraries can be screened by chemokine. Appropriatelabel techniques, e.g., anti-FLAG antibodies, will allow specificlabeling of appropriate clones.

A receptor for CCL27 and CCL28 was identified by screening a panel ofknown and orphan chemokine from human and murine sources. Apositive-screening receptor was identified by calcium flux assays, usingmouse cells transfected with the above-mentioned receptors. Thisreceptor is CCR10 (a.k.a. GPR2). Human CCL28 produced a positive signalwith mouse cells transfected with human CCR10 or with mouse CCR10 (Wang,et al. (2000) J. Biol. Chem. 275:22313-22323).

XIV. Immunohistochemical Localization

The antibody described above is used to identify expression of CCL28 orCCL27 in various tissues. Methods for immunohistochemical staining aredescribed, e.g., in Sheehan, et al. (eds.) (1987) Theory and Practice ofHistotechnology, Battelle Press, Columbus, Ohio.

XV. Contact Allergy and Psoriasis in Humans, Increase in CCL27 Levels,and Increase in Cells Bearing CCL27 Receptor

CCL27 was found in skin of healthy subjects, where it occurs in basalkeratinocytes of the basal layer, and in endothelial cells of thesuperficial dermal plexus. With nickel exposure, CCL27 levels increasedin the epidermal keratinocytes of the basal and suprabasal layers, aswell as in the dermis (unexposed skin showed only weak expression ofCCL27 in the dermis) (FIG. 2 of Homey, et al. (2002) Nature Med.8:157-165).

CCL27 receptor was not detected in the epidermis of skin from healthysubjects, though it was found in scattered cells of the superficialdermal plexus. The weak expression in the dermis of normal skincorresponded to CD31⁺ endothelial cells.

With nickel exposure, skin was invaded by lymphocytes bearing CCL27receptor, and strong CCL27 receptor signals were found in theperivascular, subepidermal, and intraepidermal regions (FIG. 2 of Homey,et al. (2002) Nature Med. 8:157-165). These results support a role forCCL27 to CCR10 interactions in modulating human skin disease states.

As mentioned above, CCL27 receptor is not readily detected in healthyepidermis. However, lesional skin from patients with psoriasis or atopicdermatitis showed strong CCL27 receptor expression, where expression waslocated on lymphocytes (FIG. 1 of Homey, et al. (2002) Nature Med.8:157-165). These results support a role for CCL27 to CCR10 interactionsin modulating human skin disease states.

XVI. Animal Models of Allergies and Treatment with Anti-CCL27 Antibodies

An animal model of contact allergy can be produced bydinitrofluorobenzene (DNF). DNF treatment provoked an increase in CCL27levels in skin, and infiltration of white blood cells, and swelling.Treatment with anti-CCL27 antibody resulted in a reduction of skinswelling. Anti-CCL27 antibodies were from R & D Systems, Minneapolis,Minn.

An animal model of chronic atopic dermatis can be produced by ovalbumintreatment. Ovalbumin treatment results in infiltration of CD4⁺lymphocytes in the skin, with some infiltration by eosinophils. CCL27 ispresent in keratinocytes of normal mice, as determined by histologicalanalysis. Ovalbumin-induced skin swelling and inflammation was reducedby anti-CCL27 antibodies, where lymphocyte recruitment was reduced by90%. Anti-CCL27 antibody seemed not to change eosinophil levels,however, demonstrating specificity of anti-CCL27 action (FIG. 6 ofHomey, et al. (2002) Nature Med. 8:157-165).

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

TABLE 2 Description of SEQ ID NOs. Sequence Identifier Sequence SEQ IDNO: 1 human CCL28 nucleic acid sequence SEQ ID NO: 2 human CCL28polypeptide sequence SEQ ID NO: 3 human CCL28 nucleic acid sequence SEQID NO: 4 human CCL28 nucleic acid sequence SEQ ID NO: 5 hCCL27 nucleicacid sequence SEQ ID NO: 6 hCCL27 amino acid sequence SEQ ID NO: 7mCCL27 nucleic acid sequence SEQ ID NO: 8 mCCL27 amino acid sequence SEQID NO: 9 human MCP-1 polypeptide sequence SEQ ID NO: 10 human MIP-3αpolypeptide sequence SEQ ID NO: 11 longer transcript with human CCL28SEQ ID NO: 12 longer polypeptide with human CCL28

1. An isolated antibody or antigen-binding fragment thereof thatspecifically binds to a polypeptide consisting of the amino acidsequence of SEQ ID NO: 6 or to an antigenic fragment comprising 10 ormore contiguous amino acids thereof.
 2. The antibody or antigen-bindingfragment of claim 1 wherein the polypeptide consists of 20 contiguousamino acids of SEQ ID NO:
 6. 3. The antibody or antigen-binding fragmentof claim 2 wherein the polypeptide consists of 30 contiguous amino acidsof SEQ ID NO:
 6. 4. The antibody or antigen-binding fragment of claim 3wherein the polypeptide consists of 50 contiguous amino acids of SEQ IDNO:
 6. 5. The antibody or antigen-binding fragment of claim 4 whereinthe polypeptide consists of 70 contiguous amino acids of SEQ ID NO: 6.6. An isolated antibody or antigen-binding fragment thereof thatspecifically binds a polypeptide consisting of the amino acid sequenceof SEQ ID NO:
 6. 7. An isolated monoclonal antibody that specificallybinds a polypeptide consisting of the amino acid sequence of SEQ ID NO:6.
 8. The antibody or antigen-binding fragment of claim 1 which is apolyclonal antibody; a monoclonal antibody; a single chain antibody; aFab fragment; an Fv fragment; a labeled antibody; a humanized antibodyor an anti-idiotypic antibody.
 9. The antibody or antigen-bindingfragment of claim 8 which is labeled with a member selected from thegroup consisting of a radiolabel, peroxidase, alkaline phosphatase,biotin, avidin and ¹²⁵I.
 10. The antibody or antigen-binding fragment ofclaim 6 which is a polyclonal antibody; a monoclonal antibody; a singlechain antibody; a Fab fragment; an Fv fragment; a labeled antibody; ahumanized antibody or an anti-idiotypic antibody.
 11. The antibody orantigen-binding fragment of claim 10 which is labeled with a memberselected from the group consisting of a radiolabel, peroxidase, alkalinephosphatase, biotin, avidin and ¹²⁵I.
 12. The antibody of claim 7 whichis a labeled antibody.
 13. The antibody of claim 12 which is labeledwith a member selected from the group consisting of a radiolabel,peroxidase, alkaline phosphatase, biotin, avidin and ¹²⁵I .
 14. Theantibody or antigen-binding fragment of claim 1 bound to saidpolypeptide.
 15. The antibody or antigen-binding fragment of claim 6bound to said polypeptide.
 16. The antibody of claim 7 bound to saidpolypeptide.
 17. The antibody or antigen-binding fragment of claim 1immobilized on a solid substrate.
 18. The antibody or antigen-bindingfragment of claim 6 immobilized on a solid substrate.
 19. The antibodyof claim 7 immobilized on a solid substrate.
 20. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragment of claim1 and a pharmaceutically acceptable carrier.
 21. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragment of claim6 and a pharmaceutically acceptable carrier.
 22. A pharmaceuticalcomposition comprising the antibody of claim 7 and a pharmaceuticallyacceptable carrier.